ReviewArticle Physiological Roles of DNA Double-Strand Breaksdownloads.hindawi.com/journals/jna/2017/6439169.pdf · ReviewArticle Physiological Roles of DNA Double-Strand Breaks

Review ArticlePhysiological Roles of DNA Double-Strand Breaks

Farhaan A Khan and Syed O Ali

School of Clinical Medicine Addenbrookersquos Hospital University of Cambridge Hills Road Cambridge CB2 0SP UK

Correspondence should be addressed to Syed O Ali soa22camacuk

Farhaan A Khan and Syed O Ali contributed equally to this work

Received 8 July 2017 Accepted 24 September 2017 Published 18 October 2017

Academic Editor Shigenori Iwai

Copyright copy 2017 Farhaan A Khan and Syed O Ali This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Genomic integrity is constantly threatened by sources of DNA damage internal and external alike Among the most cytotoxiclesions is the DNA double-strand break (DSB) which arises from the cleavage of both strands of the double helix Cells boasta considerable set of defences to both prevent and repair these breaks and drugs which derail these processes represent animportant category of anticancer therapeutics And yet bizarrely cells deploy this verymachinery for the intentional and calculateddisruption of genomic integrity harnessing potentially destructiveDSBs in delicate genetic transactionsUnder tight spatiotemporalregulation DSBs serve as a tool for genetic modification widely used across cellular biology to generate diverse functionalitiesranging from the fundamental upkeep of DNA replication transcription and the chromatin landscape to the diversification ofimmunity and the germline Growing evidence points to a role of aberrant DSB physiology in human disease and an understandingof these processes may both inform the design of new therapeutic strategies and reduce off-target effects of existing drugs Herewe review the wide-ranging roles of physiological DSBs and the emerging network of their multilateral regulation to consider howthe cell is able to harness DNA breaks as a critical biochemical tool

1 Introduction

Just as DNA breakage can devastate genomic integrity it canalso be deliberately and precisely exploited by cells in featsof genetic craftsmanship Indeed various integral cellularprocesses and genetic transactions are underpinned by themeasured use of such potentially deleterious breaks

The genome is continuously exposed to a panoply ofDNA damaging agents both endogenous and exogenouswhich pose a considerable threat to genomic integrity Thesegenotoxic insults result in diverse DNA lesions includingmismatches base adducts pyrimidine dimers intra- andinterstrand cross links DNA-protein adducts and strandbreaks [1] Among themost noxious lesions are DNA double-strand breaks (DSBs)wherein both strands of the double helixare broken through cleavage of phosphodiester linkages in thebackbone of the duplex [2] There is great variation amongDSBs both structurally and in terms of the mechanisms oftheir generation While simple DSBs such as those gener-ated by restriction endonucleases may have either blunt or

staggered ends DSBs induced by physical or chemical agentssuch as by ionising radiation and radiomimetic chemicalsboth of which are used in cancer therapy can displayincreased complexity This includes chemical modificationsof termini differing latency of generation indirect DSBformation from processing of other forms of DNA dam-age and chromatin destabilisation from clustering of DSBs[3]

DSBs are highly mutagenic and can induce potentiallytumorigenic chromosomal translocations If unrepairedDSBs may also lead to cell death [4] It is thus of utmostprimacy that such cytotoxic breaks are rapidly detectedsignalled and repaired Indeed DSBs elicit a potent DNAdamage response (DDR) comprising DNA repair cell cyclearrest andor apoptosis [2] Two major pathways operate ineukaryotic cells in the repair of endogenously or exogenouslyinduced DSBs nonhomologous end-joining (NHEJ) andhomologous recombination (HR) Unlike error-prone NHEJwhich operates throughout the cell cycle HR is essentiallyerror-free and is limited to the S and G2 phases [5]

HindawiJournal of Nucleic AcidsVolume 2017 Article ID 6439169 20 pageshttpsdoiorg10115520176439169

2 Journal of Nucleic Acids

Despite the dangers inherent in such lesions DSBs areprecisely employed for the intentional but controlled disrup-tion of genomic integrity in biological processes The cellularroles of physiological DSBs can be broadly considered asone of two major functionalities genetic recombination ormanipulation of DNA topology In the former role DSBscan act as genomic ldquoshufflersrdquo carefully but permanentlyrecombining DNA segments for genomic diversificationin lymphocytes [6 7] and germ cells [8] In contrasttopoisomerase-mediated DSBs serve as genomic ldquosculptorsrdquomodulating higher-order DNA structure and serving tofacilitate DNA replication and transcription [9ndash11] regulategene expression [12ndash15] and alter chromatin state [16 17]While this latter form of DSB may at first glance appear tobe little more than a transient intermediate these ldquocleavagecomplexesrdquo are structurally DSBs also exist as longer-livedspecies can be endogenously or exogenously converted intoabortive breaks and are highly spatiotemporally regulatedto both produce functionally diverse genomic contortionsand prevent genotoxicity or failure of genetic transactions[18 19] Hence both sources of DSB will be considered in thisdiscussion

Here we review the wide-ranging biological functions ofthese various physiologicalDSBs and themultilayered regula-tion thereof together constituting cellular ldquoDSB physiologyrdquo(Figure 1) By considering DSB physiology in its variousforms we explore how the cell is able to harness potentiallydestructive DSBs in delicate genetic operations

2 Genetic Recombination DSBs asGenomic Shufflers

21 Physiological DSBs in V(D)J Recombination The roleof physiological DSBs in the diversification of the adaptiveimmune response is well documented V(D)J recombinationdescribes the process whereby lymphoid cells recombine arepertoire of germline variable (V) diversity (D) and joining(J) exon gene segments in various permutations to generateenormous antigen-receptor diversity in immunoglobulins(Ig) and T cell receptors (TCRs) All three exon segments areused for assembling the variable region of the Ig heavy chainand the TCR 120573 and 120575 chains whereas only V and J segmentsare required for the Ig light chain and TCR 120572 and 120574 chains[20]The process involves large genomic rearrangements andDSBs act in these processes as ldquorecombinogenic biomarkersrdquospecifying the recruitment of the recombinatorial machineryIt thus follows that the spatiotemporal regulation of DSBinduction and repair is critical to the precise execution ofthis genetic shuffling and perturbations thereof can producedisease in humans The mechanism proceeds as two broadphases cleavage and joining corresponding to the processesof DSB formation and DSB resolution respectively

211 Cleavage DSB Formation Each V D and J segment isflanked by a recombination signal sequence (RSS) compris-ing conserved heptamer-spacer-nonamer sequence elements[21 22] Cleavage is initiated by the recognition of these RSSelements by the lymphoid-cell specific recombinase complex[23 24] Components of this complex RAG-1 and RAG-2

bind one RSS from each of the paired gene segments to formRAG-RSS complexes associated with each signal sequence[25 26] The two RSS groups are then approximated andsynapsis proceeds to form a precleavage synaptic complex(PcSC)ThePcSC is typically directed between gene segmentswith different RSS spacer lengths (either 12 or 23 base pairs)conforming to the 1223 restriction rule [27] PcSC forma-tion is facilitated by the DNA-bending high-mobility groupproteins HMGB1 and HMGB2 which are widely involved inchromatin architecture [28] Here HMGB12 act to enhanceRAG12 binding to and cleavage of the 23 RSS possibly viastabilisation of RAG-mediated bending of the 23 RSS spacer[29ndash33]The RAG proteins proceed to introduce a nick at thejunction between the RSS heptamer and coding sequenceWhile RAG-1 alone but not RAG-2 possesses DNA-bindingactivity and can mediate signal recognition association ofRAG-1withRAG-2 increases sequence specificity and the sta-bility of the enzyme-DNA complex [34 35] Nick formationis followed by a RAG1-mediated transesterification reactionforming a DSB comprising a blunt 51015840-phosphorylated signalend and a hairpin coding end [23 36] The DSB is thenenclosed in a postcleavage synaptic complex before NHEJrepair in the joining phase [37]

212 Joining DSB Resolution Prior to joining hairpin cod-ing ends are nicked open through the endonuclease activityof the nuclear enzyme Artemis which complexes with theserinethreonine kinase DNA-PKcs which in turn activatesArtemis by phosphorylation [38 39] However studies alsoindicate a postcleavage role for RAG proteins in openinghairpin coding ends through 31015840-flap endonuclease activity[40ndash42] as well as serving as a scaffold to shield theDNA endsfrom aberrant nuclease digestion [43] Modifications duringrecombination are introduced through Artemis-mediatedasymmetric hairpin opening andor terminal deoxynu-cleotidyl transferase- (TdT-) mediated nucleotide additionThe former mechanism enables palindromic repeat or ldquoPrdquonucleotide addition as well as nucleotide loss from codingends [44] while TdT-mediated addition inserts random ldquoNrdquonucleotides at coding ends in a 51015840-to-31015840 direction [38 45]TdT a template-independent polymerase is recruited by acomplex of NHEJ repair proteins comprising XRCC4 DNAligase IV and DNA-PK (itself a complex of Ku70 Ku86and DNA-PKcs) [46ndash49] The subsequent activity of DNApolymerase 120582 and 120583 combined with exonucleases generatescompatible coding ends [50 51] which are then ligated bythe XRCC4-DNA ligase IV-XLF complex to produce therecombined V(D)J product [46 47 52]

213 Regulation of DSBs in V(D)J Recombination An impor-tant point of control in the DSB formation step of V(D)Jrecombination is the cell cycle-dependent regulation ofRAG expression Cyclin A-CDK2 phosphorylation-mediateddegradation of RAG-2 at the G1S transition [53] exerts tem-poral control over RAG-induced DSBs by restricting RAG2expression to the G1 phase of the cell cycle [54] Further-more in p53-deficient mice a nonphosphorylatable T490Amutation of the phosphodegron in the C-terminus of RAG-2 results in increased lymphoma formation characterised by

Journal of Nucleic Acids 3

(i) Basal transcription(ii) Activity-induced DSBs

Mitochondrial DNA(i) Potential role in relief

of topological stressfor mtDNA transactions

(ii) Potential role in ageing

Chromatin architecture(i) Chromatin remodelling

(ii) Mitotic chromosomecondensation andsegregation

DNA replication(i) Resolution of supercoils

precatenanes andcatenated dimers

(ii) Prevention of fork arrest(iii) Faithful chromosome

segregation

Manipulation of DNA topologyGenomic sculptor

Lymphoid cells Germ cells

Genetic recombinationGenomic shuer

V-DJ Joining

V(D)J recombination(i) Antigen-receptor

diversification(ii) B and T lymphocyte

selection

Class switchrecombination

(i) Ig isotypeswitching

(ii) Effector function specification

Meioticrecombination

(i) Germlinediversification

(ii) Accuratechromosomesegregation

Physiological DSBs

DNA

RNA

RNA polymerase

3

5

3

5

3

5

tor

hocytyy e

isotypyy ewitchingffeffff ctor fuff nctionecification

Transcription

Figure 1 Summary of the diverse roles of physiological DSBs in biological processes

an increased frequency of clonal chromosomal translocations[55] illustrating the importance of this regulation for theintegrity of recombination

Additional temporal control is exerted over RSS selectionfor DSB formation and only certain RSS sites are availablefor recombination exhibiting cell- and stage-specific biases[56 57] The great accuracy of the recombinatorial machin-ery both in RSS specification and in avoiding inauthentic

RSS-like (cryptic RSS cRSS) sites which occur around onceper 600 bases in random DNA sequences [58] involvescontrolling the accessibility of the recombinase to its substratefor DSB generation The discovery of the concurrence of IgHvariable region (VH) germline transcription with its joiningwith DJH suggested an open chromatin state was requiredfor recombination [57] This ldquoaccessibility hypothesisrdquo is nowthought to involve chromatin architectural changes brought


about by DNA and epigenetic modifications chromatin-binding proteins and cis-acting enhancer elements Theseultimately produce a transcriptionally active locus charac-terised by DNA hypomethylation activating histone mod-ifications DNase I sensitivity and increased RNA poly-merase II occupancy [59ndash64] RAG2 has been shown tospecifically recognise the histone modification H3K4me3 amarker enriched at active promoters via its PHD finger aninteraction which also stimulates RAG complex activity [6566]This recognition likely serves to localise the recombinaseto target RSSs by active promoters and upregulate recombi-nation thereat [64] Modulation of chromatin packing alsodetermines the order of rearrangement observed in B cell Ig(heavy chain before light chain) and T cell TCR (120573 chainbefore 120572 chain) as well as the process of allelic exclusion [56]Aside frommodulation of accessibility of RSS sites to bindingand cleavage by the recombination machinery target locusposition within chromatin compartments of the nucleus andthe structure of the locus itself are also directed to facilitaterecombination [64]

214 Dysregulation of DSB Physiology in V(D)J Recombi-nation Broadly perturbations in V(D)J recombination canbe from either an inability to form DSBs an inability torepair them or inaccurate pairing of RSSs Although viableRag-1- or Rag-2-deficient mice are unable to carry outV(D)J recombination and lack mature B and T lymphocytespresenting with severe combined immune deficiency (SCID)[67 68] Missense mutations in either RAG-1 or RAG-2 genesare also observed in humans with an analogous form ofautosomal recessive SCID known as Omenn syndrome [69]Genetic studies of polymorphisms in wild-type and Omennsyndrome patients as well as biochemical data characterisingrecombinatorial competence of Omenn syndrome mutantssuggest that RAG-1 and RAG-2 mutants exhibit lower effi-ciency of V(D)J recombination [69] These mutations areassociated with decreased RAG DNA-binding and cleavageactivity and a lower efficiency of interaction between RAGproteins [69]

Impairment of the joining phase of V(D)J recombinationcan also cause immunodeficiency While all mammalianNHEJ pathway mutants display sensitivity to ionising radi-ation and a lack of B and T lymphocytes a spectrum ofphenotypes is observed in mouse models DNA-PKcs andArtemis mutants exhibit subtle defects in DSB repair [70]but display no growth retardation [71] Hairpin opening islost in Artemis-deficient SCID [72] andDCLRE1C hypomor-phism in humans has been found to underlie an Omennsyndrome phenotype associated with defective endonucle-ase activity [73] Ku70 and Ku80 mutants display similarphenotypes of radiosensitivity and growth retardation [7475] likely due to the interdependence conferred by theirheterodimerisation [76] Mutants of the critical end-joiningproteins XRCC4 and DNA ligase IV have the most severephenotype both displaying V(D)J recombination defectsimpaired lymphocyte development p53-dependent neuronalapoptosis and embryonic lethality [77ndash79] These findingsthus point towards a crucial role of DSB repair in BT cellphysiology and adaptive immune diversification

Lymphomas have been found to exhibit a range of chro-mosomal aberrations as a result of erroneous DSB handlingin V(D)J recombination Oncogenic lesions have been foundto include chromosomal translocations insertions deletionsand inversions and broadly result from errors in RSS pairingor joining [80ndash82] Inaccurate RSS recognition can produceRSScRSS pairings which may exert tumorigenic effects viaamplification of a protooncogene or juxtaposition of tran-scriptional regulatory elements from the antigen-receptorlocus with a protooncogene driving dysregulated expression[6] cRSScRSS pairings may also arise which can producetranslocations or deletions [6] Aberrant DSB formationhas also been found to underlie the reciprocal (t14 18)translocation found in 90 of follicular lymphomas [83 84]In this case it has been shown that cleavage of non-B-formDNA at a non-RSS ldquotranslocation fragile zonerdquo called themajor breakpoint region (MBR) at the Bcl-2 gene producesa translocation with the DSB at the Ig heavy chain locuson chromosome 14 [84] Recent work on minichromosomalsubstrates has revealed the importance of CpGmethylation inthe localisation of aberrant DSB formation at the Bcl-2 MBRand that breakage is AID-dependent (activation-inducedcytidine deaminase) [85] Lymphomas have also been foundto exhibit defects in DSB joining producing chromosometranslocations by the ldquoend donationrdquomodelThis involves theerroneous joining of a RAG-inducedDSB at an authentic RSSwith a non-RAG-mediated DSB [86]

22 Physiological DSBs in Class Switch Recombination Classswitch recombination (CSR) is another example of a DSB-dependent recombination event occurring in mature B cellsIn response to antigenic stimuli and costimulatory signalsfrom other immune cells B cells undergo CSR to alter theirIg constant heavy chain while retaining the same antigenspecificity in effect generating an alternative Ig isotypewith the same paratope but distinct effector functions [87]This enables a permanent switch of expression from thedefault IgM and IgD to alternative Ig isotypes (IgA IgGor IgE) by recombination of the heavy chain (IgH) locusRecombination is achieved through cleavage and excisionof constant heavy chain (CH) genes via DSB generationfollowed by ligation of the remaining segments through DSBresolution [87]

Conserved motifs called switch (S) regions upstreamof each CH gene are the site of DSB generation [88 89]AID initiates recombination by conversion of multiple Sregion deoxycytidine residues into deoxyuracil [90ndash93]The resulting UG mismatch can be converted into a DSBthrough base-excision repair (BER) andor mismatch repair(MMR) DNA repair pathways [87] BER proceeds throughexcision of deoxyuracil by uracil DNA glycosylase (UNG)[93 94] followed by nicking of the resulting abasic site byapurinicapyrimidinic endonucleases (APE) [87 95] AID-mediated deamination on the opposite strand introduces asecondnick in close proximity thereby generating a staggeredDSB [89 96] Processing of these overhangs forms blunt freeDNA ends which are permissive for shuffling [87 97]

As aforementioned MMR is another pathway employedin CSR DSB generation and is thought to provide auxiliary


support to BER-mediated mismatch excision [98] Indeedmultiple studies ofMMR-deficientmurine B cells have shownimpaired and aberrant CSR implicatingMMRproteinsMsh2Msh6 Mlh1 Pms2 and Exo1 in the recombination process[99ndash104] Onemodel proposes that while the aforementionedBER-induced SSBs can automatically form DSBs if near oneanother distant SSBs within the large S regions can beconverted into DSBs by assistance from MMR [105] Thisfirst involves recognition of an AID-induced UG mismatchby a heterodimer of Msh2-Msh6 followed by recruitmentof the endonuclease heterodimerMlh1-Pms2These proteinsin turn recruit the 51015840-31015840 exonuclease Exo1 to a nearby BER-induced 51015840 SSB and Exo1 proceeds to excise the strandtowards the mismatch and beyond until it reaches anotherSSB thus producing aDSB [105] Alternatively it has also beenproposed that if Exo1-generated single-stranded DNA gapsare formed on opposite strands this too may form a DSB[106]

After intrachromosomal deletion of the interveningunwanted CH genes the free end of the variable domainregion is ligated to that of the new constant domain exon bythe NHEJ machinery [107ndash110]

221 Regulation of DSBs in CSR Recombination underlyingisotype switching of constant heavy chain genes is regulatedby germline transcripts which modify switch region accessi-bility to the DNA handling machinery [105] Transcriptionbeginning at germline promoters upstream of a target Sregion generates a sterile (noncoding) transcript known asa germline transcript (GT) [111 112] Activation of specificcytokine-inducible transcription factors and their action atgermline promoters determines which GT is generated [113]These germline transcripts direct AID to specific S regionsthus spatially regulating DSB generation and in turn CSR[105 114]

AID deamination can only occur on single-strandedDNA (ssDNA) [115] and one proposed mechanism of GT-mediated regulation is optimisation of S region structure forAID recognition [115] This could be achieved through thecreation of short ssDNA tracts in the S regions parallelingsomatic hypermutation [116] or through RNA-DNA hybridR-loop formation [117 118] AID can also be directly recruitedto RNA Pol II at the initiation or elongation phase of GTformation which may facilitate AID targeting [105 119]Further regulation may also occur at the level of higher-order chromatin structure whereby histone modifications inthe transcribed region may modulate AID accessibility [120]While the exact mechanism of this architectural control isunclear it appears to be associated with B cell activation AIDexpression and RNA Pol II enrichment [120 121] It is thusclear that various levels of regulation act to spatiotemporallyrestrain DSB formation and repair in CSR

23 Physiological DSBs in Meiotic Recombination Beyond itsroles in lymphoid cells DSB-induced recombination also actsinmeiosis where it is essential for both the fidelity of chromo-some segregation and diversification of the germline Meiosisis a form of reductive cell division exclusive to gametogenesisand involves two successive divisions following replication

known as MI andMII [122] A hallmark of meiosis is meioticrecombination between homologous chromosome pairs ordyads which is reliant on DSB induction and HR repairresulting in exchange of genetic information between non-sister chromatids [122] These ldquocrossoverrdquo events essentiallyproduce shuffling of alleles in each chromatid giving riseto a unique haploid complement in the resultant gametesultimately creating genetic diversity within the populationFurthermore meiotic recombination establishes physicallinkages between homologs allowing accurate segregation bythe spindle apparatus [123] Just as in V(D)J recombinationand CSR careful spatiotemporal regulation of DNA breakinduction and resolution is critical to the integrity of theprocess

231 DSB Formation Meiotic recombination is initiatedby DSB induction by a dimer of the topoisomerase-liketransesterase Spo11 which acts via a covalently linked proteintyrosyl-DNA intermediate complex [124ndash127] Interestinglythe human SPO11 homolog maps to position 20q132-q133a region known to be amplified in some breast and ovariantumours possibly reflecting genomic instability due to dys-regulation of DSB formation [128] Spo11 action is criticalfor gametogenesis and loss in mice produces infertility andresults in defective meiotic recombination DSB formationand synapsis in Spo11minusminus mouse spermatocytes [129] DSBformation is also dependent on an extensive protein-proteininteraction network most studied in S cerevisiae betweenSpo11 and other meiotic proteins These proteins includecomponents of the Mre11-Rad50-Xrs2 (MRX) complex andmultiple Spo11-accessory proteins including Mei4 Mer2Rec102 Rec104 Rec114 and Ski8 which facilitate DSB for-mation [126 130] The functions of these various proteinsare incompletely understood however the WD repeat pro-tein Ski8 has been found to stabilise association of Spo11on meiotic chromatin and promotes recruitment of otheraccessory proteins [130] and Rec102 and Rec104 are known toform a multiprotein complex with Spo11 and act with Rec114to regulate Spo11 self-association [131ndash133] Rec 114 Mer2and Mei4 (RMM) constitute a separate subcomplex [134]which plays a role in the spatiotemporal regulation of DSBformation as will be discussed below

232 DSB Resolution The resulting DSBs then undergoendonucleolytic processing which releases Spo11 to allowrepair [135] This initial DSB resection is coordinated withcell cycle progression and is dependent on the activity ofthe multisubunit nuclease MRX complex and Sae2 pro-tein in budding yeast [126 136ndash138] The former complexalso acts to facilitate HR independently of its nucleolyticactivity [139] Analogous to the MRX complex the MRE11-RAD50-NBS1 (MRN) complex possesses a similar role inSPO11 removal in other eukaryotes [140] Spo11 removal isfollowed by extension of the end resection involving thehelicase Sgs1 and nucleases Exo1 and Dna2 forming 31015840single-stranded overhangs for HR [138 141] The overhangsthen undergo homology search and strand invasion catal-ysed by the strand exchange proteins Rad51 and meiosis-specific Dmc1 alongside various other cofactors [142 143]


The resulting interactions are processed to form recombi-nation products with either noncrossovers or crossoversdefined respectively as gene conversion or reciprocal geneticexchange between homologous chromosomes [142] Duringthis pairing or synapsis of dyads a proteinaceous structurecalled the synaptonemal complex establishes a zipper-likeconnection between homologous chromosomes [144] Recip-rocal interhomolog recombination results in the formation ofphysical linkages between homologous chromosomes knownas chiasmata which aid coalignment of the resultant bivalentsalong the spindle axis for accurate segregation [145]

24 Regulation of DSBs in Meiotic Recombination A multi-tude of interacting mechanisms operate in meiotic recombi-nation to control DSB formation and resolution This tightspatiotemporal regulation of DSB handling allows the inten-tional disruption of gamete DNA integrity for diversificationwhile ensuring coordinated cell cycling and ultimately gen-eration of an intact haploid germline

241 Temporal Control of Meiotic DSBs DSB induction ismechanistically coupled to DNA replication [146] and is sub-ject to strict temporal regulation [147ndash149] occurring onlybetween replication and chromosome segregation [150] Thisensures both coordinated chiasma formation and effectivehomology search for synapsis [150] In addition a multi-tude of checkpoints operate to couple DSB induction andresolution to the cell cycle to protect against the deleteriouseffects of unrepaired DSBs and also act to arrest or eliminateaberrant cells [151ndash154]

Posttranslational modifications on the Spo11-accessoryprotein Mer2 have been shown to contribute to the timelyexecution of DSB formation in meiotic recombination Phos-phorylation of Mer2 by the budding yeast cyclin-dependentkinase Cdc28 stimulates DSB formation via promotion ofinteractions both with itself and with other proteins involvedin DSB generation including Mei4 Rec114 and Xrs2 [155]It is known that Cdc28-Clb5 (CDK-S) activity increasesduring premeiotic replication and peaks before MI [156 157]and thus may help orchestrate a concerted progression ofmeiotic recombination and prophase I [155] Mer2 is alsophosphorylated by Cdc7 kinase in S cerevisiae to controlSpo11 loading on DSB target sites [158] It has been foundthat CDK-S phosphorylation on Mer2 S30 promotes DDK(Cdc7-Dbf4)-dependent phosphorylation at the adjacent S29[157] As well as being involved in meiotic recombinationCDK-S and DDK are both required for premeiotic S phase[156 159] which may contribute to coordination therewithFurthermore it has been found that the level of DDK activityrequired for premeiotic S phase is lower than that supportingits postreplicative role in meiosis [159] It has been proposedthat different thresholds for both CDK-S and DDK in theseprocesses may ensure replication precedes DSB formation[160]

Additional temporal control is afforded by meiosis-specific expression of Spo11 and accessory proteins Rec102Rec104 Rec114 andMei4 largely achieved via transcriptionalregulation [150] In contrastMer2 has been found to undergomeiosis-specific posttranscriptional regulation by a splicing

factor Mer1 which itself is only expressed in meiosis [161162] Although Mer2 transcription does occur in mitosisefficient splicing by Mer1 to generate functional Mer2 isexclusive to meiosis [161]

242 Spatial Control of Meiotic DSBs Meiotic recombi-nation and DSB formation exhibit nonrandom patterningacross the genome being more frequently localised to par-ticular sites on chromosomes called ldquohotspotsrdquo [163 164]While exchange usually takes place between alleles homolo-gous recombination between nonallelic sequences with highsequence identity may occur when DSBs form in repetitiveDNA sequences such as transposable elements and low-copy repeatssegmental duplications leading to genomicrearrangements [165] In addition it has been shown thatDSBs formed in pericentromeric regions are associated withdisruption of sister chromatid cohesion leading to misseg-regation and aneuploidy [166] Therefore the location ofDSBs and subsequent recombination is controlled for themaintenance of genomic integrity

In mice recombination hotspots have been found tobe enriched for H3K4me3 histone modifications [167 168]These hotspots are determined by the DNA-binding histoneH3K4 trimethyltransferase PRDM9 which has been foundto regulate activation of recombination loci and is a keyfactor in specification of meiotic DSB distribution [169 170]Indeed Prdm9-deficiency results in infertility and disruptionof early meiotic progression in mouse models [171] PRDM9has been shown to directly and specifically bind a 13-merDNAmotif enriched at hotspots via its C2H2-type zinc fingerarray [169 172 173] and changes in the array affect H3K4me3enrichment hotspot recombination and crossover patterns[173] Furthermore sequence variation in PRDM9 mayunderlie the intra- and interspecies differences observed inrecombination hotspot distribution [169 174] While theexact mechanism linking hotspot specification with targetingof the DSB machinery is unclear it has been proposed thatPRDM9 may recruit Spo11 itself or via a partner proteinor may promote chromatin remodelling permissive for Spo11binding [173]

Negative feedback systems acting both in cis and in transalso exert an inhibitory effect from one DSB on further breakformation thus limiting DSB frequency [8] In additionDSB formation occurs at unsynapsed chromosome segmentsimplicating interhomolog interactions in the control ofDSB site and numbers [8 175] Recombination exhibits aninterhomolog bias as opposed to occurring between sisterchromatids [176] and it has recently been shown that totalDSB number itself is a determinant of DSB repair pathwayselection and development of biased recombination [177]

DSB formation is also subject to spatial regulation viacontrol of chromosome ultrastructure During the leptotenestage of prophase I DSB formation occurs alongside thedevelopment of the axial element a proteinaceous axis com-prising various proteins including cohesin [145] The sisterchromatids form linear arrays of chromatin loops which areconnected at their bases by the axial element which is theprecursor of the lateral element of the synaptonemal complexat pachytene [145] Sites of DSB formation are found to map


to loop sequences which are ldquotetheredrdquo to the axis duringrecombination in ldquotethered loop-axis complexesrdquo [178] ChIP-chip studies in S cerevisiaehave shown that Rec114Mer2 andMei4 stably associate with chromosome axis association sitesbetween loops in a manner dependent on components of theaxial element [179]Meiotic cohesinwas found to controlDSBformation and axial element protein and RMM recruitmentat a subset of chromatin domains while cohesin-independentDSB formation was observed in other domains [179] Suchdifferences in DSBmachinery deposition between chromatindomains may contribute to the establishment of differentrecombination proficiencies potentially providing a form ofspatial regulation of DSB induction [179] Furthermore thisinteraction of RMM with axis association sites is dependenton Mer2 phosphorylation by Cdc28 which likely affordsadditional temporal coordination of DSB formation with theend of premeiotic replication [179]

3 Manipulation of DNA Topology DSBs asGenomic Sculptors

31 Physiological DSBs in the Relief of Topological Stress Toaccess the information stored in compacted nuclear DNAin such processes as transcription replication and repairrequires the repeated tangling and untangling of the DNAdouble helix Such molecular contortions introduce topolog-ical entanglements into the duplex which if left uncheckedcould compromise genomic stability [180 181] Relief ofsuch topological strain is achieved by a family of essentialenzymes the type I and type II topoisomerases The formergenerate transient single-strand breaks (SSBs) while the latterfamily hydrolyse ATP to cut the DNA double helix andgenerate a transient DSBThe type II enzyme then passes oneDNA duplex through the gap and the gap is subsequentlyresealed [182] Type II topoisomerase (Top II) activity inmammals is mediated by two isoenzymes Top IIa and TopIIb which exhibit considerable homology except in theirless conserved C-termini [183 184] and display differentsubcellular distributions during the cell cycle [185 186]Studies on Top IIab chimeric ldquotail swaprdquo proteins and C-terminal truncations have shown that the C-terminal regionscontribute to isoform-specific functionality and may mod-ulate decatenation activity [187 188] Immunohistochemicalstudies of Top II isoform expression patterns in normal andneoplastic human tissues show that Top IIa expression islargely restricted to normal proliferative tissue and is highlyexpressed in certain tumour types In contrast Top IIb iswidely expressed across both proliferative and quiescent cellpopulations although at a higher level in normal proliferativetissue and tumours [189 190]

While in general a Top II-induced DSB is resolvedin the enzymersquos catalytic cycle it does possess a capacityfor formation of abortive breaks [19] and can form longer-lived complexes in certain functions [12 191] and bothinappropriately low or excessive formation of DSB interme-diates can compromise cellular viability thus necessitatingcommensurate regulation [18] The critical involvement ofDNA topoisomerases in various essential cellular processeshas led to the development of topoisomerase poisons for use

as antibacterial [192 193] or anticancer therapeutics [194 195]While catalytic inhibitors block enzymatic activity withouttrapping of the covalently linked DNA-enzyme intermediatecleavage complex the majority of clinically used Top II-targeting drugs act as Top II poisons accumulating com-plexes by either blocking religation or increasing complexformation The resultant DSBs drive mitotic arrest andapoptotic cell death illustrating the toxicity of uncontrolledDSB physiology [196] The tight spatiotemporal regulation ofTop II-mediated DSB formation enables the measured usageof these potentially deleteriousDNAbreaks as a physiologicaltool for the relief of topological stress

311 Type II Topoisomerase DSB Handling Supercoilingentanglement and knotting are frequent topological obsta-cles encountered in DNA physiology Type II DNA topoi-somerases through DSB induction and repair act to re-solve such obstacles using a repertoire of supercoil in-troductionrelaxation catenationdecatenation and knot-tingunknotting processes [197 198] Eukaryotic Top IIenzymes are homodimeric proteins with symmetricallypaired domains whereof each interface serves as a ldquogaterdquo tocontrol the passage of DNA strands [180] While catalyticefficiency is a function of the structural interplay betweenthe DNA substrate and the type II topoisomerase isoenzymethe general mechanism is conserved across the enzymefamily The overall cycle acts to relax DNA involving DSBinduction strand passage and DSB repair and proceeds viaa phosphotyrosine-linked DNA-enzyme intermediate [199]The aforementioned DSB-dependent relief of DNA topolog-ical tension is of particular importance in the processes ofDNA replication transcription and compaction into higher-order structures

32 Physiological DSBs in DNA Replication Replication forkarrest such as due to impassable DNA damage can give riseto DSBs [200 201] which are highly mutagenic and cytotoxicand elicit a potent DDR comprising HR-mediated repair cellcycle arrest and possibly apoptosis to ensure the faithfulinheritance of genetic information [202ndash204] Indeed drugswhich promoteDSB formation or target DSB repair representan important category of chemotherapeutics for cancer [205206] In spite of all this Top II-induced DSBs play an integralrole in replication illustrating how the careful regulation ofa potentially destructive lesion can be exploited for cellularphysiology

Top II enzymes are essential in the elongation of repli-cating DNA chains and during segregation of newly repli-cated chromosomes During semiconservative replicationthe replisomal machinery at the advancing replication forkforces the intertwined DNA strands ahead of it to becomeoverwound or positively supercoiled These positive super-coils are redistributed behind the fork by the rotation ofthe replicative machinery around the helical axis of theparental duplex This causes intertwinement of the newlyreplicated DNA double helices forming precatenanes [197]Studies in yeast reveal a necessity of either Top I or TopII in elongation acting as replication ldquoswivelsrdquo to relaxpositive supercoiling and resolve precatenanes respectively


[9 207ndash209] As replication nears completion the unrepli-cated double-stranded DNA (dsDNA) segment in betweenthe converging forks becomes too short for the action of atype I topoisomerase and replication must first be completedresulting in intertwinement of the replicated duplexes intocatenated dimers [208 210] These intertwines are decate-nated by the action of Top II to allow for chromosomeseparation inmitosis [211 212] In yeast which only possessesa single type II topoisomerase loss of the enzyme resultsin chromosome nondisjunction accumulation of catenateddimers and inviability at mitosis [211 213 214] In contrast ithas been shown in human cells that Top IIa but not Top IIb isessential for chromosome segregation [215]

33 Physiological DSBs in Transcription Transcription ofnascent mRNA presents topological obstacles akin to thoseseen during replication [216] Transcription elongation isaccompanied by changes in the local supercoiled state ofDNA forming positive supercoils ahead and negative super-coils behind the transcriptional machinery [197 198 216217] While loss of Top I or Top II alone in yeast generallydoes not compromise transcription top1 top2 temperature-sensitive double mutants exhibit significant inhibition ofrRNA synthesis and to a lesser extent mRNA synthesisat nonpermissive temperatures [207] In addition doublemutants accumulate negative supercoils in extrachromoso-mal plasmids which appear to promote transcription initi-ation of ribosomal minigenes on such plasmids [218 219]More recent studies of yeast rRNA synthesis suggest distinctroles of Top II and Top I acting respectively to relievepositive supercoiling ahead of and negative supercoilingbehind the advancing polymerase [11] Pharmacological inhi-bition of Top II in human cells is found to decrease PolI transcription and Top IIa depletion both impairs Pol Ipreinitiation complex (PIC) formation and produces a lossof DSBs at these sites It has thus been proposed that Top IIa-induced DSBs are involved in topological changes at rDNApromoters to facilitate PIC formation [220]

As aforementioned top2 yeast exhibits normal levels oftranscription however it has been shown in budding yeastthat loss of Top2 produces a specific reduction in Pol II-dependent transcription of genes longer than around 3 kbattributed to defective elongation due to Pol II stalling [221]More recently Top II-induced DSBs have been proposedto act in a mechanism involving the transcription factorTRIM28 andDNAdamage signalling to promote Pol II pauserelease and transcription elongation [222]

331 Activity-Induced DSBs in Transcription RegulationTranscription has long been known to stimulate HR in alocus-specificmanner [223ndash225] and it is emerging that phys-iological DSBs may be involved in regulated transcription[191 222] Furthermore it has recently been found that DSBsinduced at transcriptionally active sites in human cells prefer-entially promote HR over NHEJ and this repair is dependenton the transcription elongation-associated epigenetic markerH3K36me3 [226] It is thus becoming increasingly clearthat transcription and HR-dependent DSB repair may bereciprocally regulated Of particular interest is the emerging

role of so-called ldquoactivity-induced DSBsrdquo in transcriptionalcontrol adding additional complexity to the integration ofenvironmental stimuli and cell state to the modulation oftranscriptional programmes Indeed Top IIb has previouslybeen shown to be required for 17120573-estradiol-dependenttransactivation via the regulated formation of site-specificlonger-lived DSBs in target gene promoters [191] Top IIb-mediated DSB formation in a target promoter has also beenshown to be required for glucocorticoid receptor-dependenttransactivation [227] More recently the spotlight has shiftedto the broadening role of activity-induced DSBs in neuronalphysiology and neuropathology

332 Role of Activity-Induced DSBs in Neuronal FunctionNeuronal early response genes or ldquoimmediate early genesrdquo(nIEGs) are a subset of activity-regulated genes induced withshortest latency following neuronal stimulation and typi-cally encode transcription factors involved in transcriptionalmodulation for synaptic plasticity [228 229] Treatment ofneuronal cells with etoposide a Top II inhibitor whichinduces Top II-mediated DSBs [230] has been found to pro-mote upregulation of a subset of nIEGs including Fos FosBand Npas4 [12] This increase was not observed with otherDSB-inducing agents including neocarzinostatin bleomycinand olaparib suggesting a Top II-mediated mechanism [12]Furthermore etoposide-induced upregulation of Fos andNpas4 is not affected by inhibition ofDSB signalling followingATM blockade which also suggests that nIEG induction isindependent of the effects of a generic DDR to nonspecificDSBs [12] Similar to etoposide treatment CRISPR-Cas9-induced DSBs targeted to the Fos and Npas4 promoters alsoproduced upregulation thereof in the absence of neuronalactivity Together these indicate a role of DSBs acting withintarget gene promoters in the regulation of nIEG transcription[12]

Furthermore electrical or pharmacological stimulationof cultured primary neurons revealed upregulation of Fosand Npas4 mRNA accompanied by elevated 120574H2AX levels[12] a known biomarker of DSB formation [231] suggestingthe possible involvement of ldquoactivity-induced DSBsrdquo in nIEGexpression Genome-wide ChIP-seq of 120574H2AX followingstimulation with NMDA (N-methyl-D-aspartate) demon-strated preferential clustering of yH2AX elevations at activelytranscribed genes and downstream sequences Differentialpeak calling on NMDA-treated samples showed that yH2AXenrichment was confined to only 21 loci including the nIEGsFos FosB Npas4 Egr1 Nr4a1 and Nr4a3 among othertranscription factors and noncoding RNAs pointing to thespecific localisation of DSB induction following neuronalstimulation [12] Interestingly 120574H2AX was also observed toincrease following pharmacological or electrical stimulationof mouse acute hippocampal slices as well as in hippocampallysates from mice exposed to a fear-conditioning paradigm[12] This corroborates recent evidence supporting DSBinduction in mouse neurons in response to environmentalstimuli in the form of spatial exploration [232] These DSBswere found to predominate in the dentate gyrus suggest-ing a physiological role in spatial learning and memory[232] Overall these results suggest that neuronal activity


specifically induces targeted DSBs involved in transcriptionalregulation of nIEGs Interestingly studies of repair kineticsof DSBs generated in the Fos promoter following NMDAtreatment demonstrated long-lived breaks being repairedwithin 2 hours of stimulation However the significance ofthis extended lifespan is as yet unclear [12]

Other experiments showed that short hairpin RNA-mediated knockdown of Top2b in cultured primary neuronsreduces yH2AX enrichment at nIEG exons following NMDAstimulation pointing towards a role of Top IIb in activity-induced DSB formation at nIEGs [12] In ChIP-seq experi-ments genome-wide binding of Top IIb was found to almostquintuple following NMDA treatment and it was observedthat Topo IIb enrichment clustered alongside yH2AX sig-nals in both unstimulated and NMDA-treated cells [12] Inaddition yH2AX-enrichment following NMDA stimulationand etoposide treatment were strongly correlated lendingfurther support to a model of Top IIb-generated activity-inducedDSBs [12] Linking Top IIb-mediatedDSB formationwith activity-dependent nIEG transcription it was foundthat Top2b knockdown in cultured primary neurons inhibitsinduction of nIEGs following NMDA stimulation but couldbe rescued by Cas9-generated DSBs targeted to the nIEGpromoter [12]

While the mechanism whereby Top IIb-mediated DSBscould induce nIEG transcription is as yet unclear onemodel proposed involves a functional interaction with thearchitectural protein CTCF [12] CTCF organises genomicsuperstructure defining chromatin architecture to controllong-range interactions between different genomic lociMajor effects of CTCF action include the establishmentof chromatin domains and blockade of promoter-enhancerinteractions [233] Searches for motif enrichment at TopIIb binding sites have revealed greatest clustering of CTCFunder both basal conditions and NMDA treatment [12]and Top IIb itself is found to concentrate within CTCFmotifs Furthermore Top IIb is found to coimmunopre-cipitate with CTCF both under basal conditions and to agreater extent with NMDA stimulation Genome-wide CTCFbinding distribution also corresponds well with yH2AXsignals at NMDA-stimulated activity-induced DSBs and fol-lowing etoposide [12] Combining these results Madabhushiet al [12] propose a model whereby neuronal activity drivesTop IIb-induced DSB formation which relieves CTCF-basedtopological impediments to nIEG transcription While arecent proteomic analysis of the Top IIb proximal proteininteraction network revealed interaction of Top IIb withCTCF at topologically associating domain (TAD) boundaries[234] the precisemechanismwhereby activity-inducedDSBsmay overcome CTCF-generated obstacles is unknown It wasrecently postulated that activity-induced DSBs in nIEGs leadto genomic rearrangements involving transposable elementsproducing a distinct form of somatic mosaicism in neu-rons with implications on plasticity network formation andpotential interplay with age-related epigenetic changes anddisease [235]

Additional mechanistic complexity derives from the needfor concerted repair of activity-induced DSBs Top IIbexhibits preferential binding and cleavage at the promoters

of target nIEGs cooccupying the site with the transcriptionalactivator ELK1 and histone deacetylase HDAC2 the latterrepressing nIEG expression [236] Also found enriched atthese sites is the tyrosyl-DNA phosphodiesterase TDP2which acts via the NHEJ pathway to repair abortive Top IIb-induced DSBs [19 237 238] suggesting coordinated resolu-tion of such physiological breaks [12] Furthermore TDP2-deficiency in human cells produces hypersensitivity to Top II-induced DSBs and reduces Top II-dependent transcriptionillustrating the importance of regulated break repair in DSBphysiology [19]

333 Dysregulation of Physiological DSBs in NeuropathologyDefective DSB repair andor loss of DSB repair componentsis a feature of various neuropathologies and age-associatedneurodegenerative disorders including ataxia-telangiectasia(ATM) ataxia-telangiectasia-like disorder (MRE11) Nijme-gen breakage syndrome (NBS1) and Alzheimerrsquos disease[239ndash245] In addition Ercc1Δminus mice which have compro-mised nucleotide excision repair interstrand crosslink repairand DSB repair pathways display age-associated neurode-generation and progressive loss of motor function [246 247]

Increased incidence of DNA strand breakage has beenidentified in the brains of Alzheimerrsquos disease (AD) patients[248 249] andADmousemodels exhibit an elevated baselinefrequency of neuronal DSBs [232] Furthermore amyloid-120573(A120573) aggregates of which are widely recognised as a key fac-tor in AD pathogenesis is found to increase DSB formationin primary neuronal cultures via dysregulation of synaptictransmission [232 250] Expression of BRCA1 an importantHR DSB repair factor [251 252] is found to be reducedin AD brains and exhibits neuronal activity-dependent reg-ulation as well as downregulation by A120573 oligomers [253]Moreover short hairpin RNA knockdown of BRCA1 inwild-type mice impairs repair of activity-induced DSBs[253]

There is growing evidence suggesting that aberrant TopII activity may play a role in the aetiology of neurodevelop-mental defects Recent work involving clinical whole-exomesequencing identified a novel TOP2B mutation associatedwith intellectual disability autistic traits microcephaly anddevelopmental retardation [254] and homozygous mutationsin TDP2 have been linked with cases of intellectual disabilityepilepsy and ataxia [19] Top IIb has been shown to beimportant in neuronal differentiation and survival and isupregulated during the transition from mouse embryonicstem cells to postmitotic neurons with reciprocal downregu-lation of Top IIa Furthermore loss of Top IIb in mice resultsin premature degeneration and apoptosis of neurons at laterstages of differentiation concomitant with transcriptionalderegulation of genes involved in neurogenesis and celldivision [13 14] Pharmacological inhibition of Top I inmouse cortical neurons results in preferential downregula-tion of long genes many of which are associated with autismspectrum disorder As Top IIab inhibition by ICRF-193produces a similar length-dependent transcriptional reduc-tion it is possible that Top II is also responsible for theregulation of certain autism-associated candidate genes [255]Furthermore it has been proposed that environmental and


dietary sources of topoisomerase poisons and inhibitors inearly neurodevelopment could be a potential cause of autismand there have been calls to factor this into food safety andrisk assessments [256 257]

Together these findings point towards the importance ofDSB repair regulation in neuronal physiology and activity-induced DSB handling but further studies will be requiredto elucidate more specific roles of activity-induced DSBs inneuropathology

34 Physiological DSBs in Chromatin Architecture Top II isa highly abundant chromatin protein constituting 1-2 oftotal chromosomal protein content in mitosis [258] Studiesof isolated chromosomes reveal association of one Top IImolecule per 23000 base pairs of DNA largely at andaround loop bases suggesting a structural role in higher-order DNA architecture [258 259] Indeed Top II bindingand DSB formation sites have been identified in matrix-attachment regions cis-elements which are involved in long-range chromatin organisation [260 261] However photo-bleaching experiments have revealed both Top II isoforms tobe highly mobile in the chromosomal scaffold arguing for amore complex dynamic interaction with chromatin structure[262]

DNA topology and chromatin structural changes areknown to be intricately related [263] Early work withtemperature-sensitive top2 yeast [212] and complementa-tion experiments with Top II-immunodepleted Xenopus eggextracts [264] revealed Top II to be important in chromo-some condensation and segregation in mitosis presagingthe discovery of a more active role of the enzyme in chro-matin remodelling Indeed Top II has been identified asa component of the Drosophila ATP-dependent chromatinremodelling complex CHRAC (chromatin-accessibility com-plex) [265] and mammalian BAF chromatin remodellingcomplexes are found to promote Top IIa activity [266]Furthermore Top II-mediated DNA supercoil relaxation isstimulated by the Drosophila protein Barren [267] whichis involved in chromosome condensation and disjunction[268] ChIP experiments in S pombe demonstrate an inter-genic localisation of both Top I and Top II which stronglycorrelates with distributions of the chromodomain ATPaseHrp1 and the histone chaperoneNap1 both involved in nucle-osome disassembly [269ndash271] Top II-dependent chromatinremodelling is also important in apoptotic chromosome con-densation and caspase-activated DNase (CAD) associationis found to promote Top IIa activity [272] While the exactmechanistic role of DSB handling in chromatin packing isunclear AFM and fluorescence imaging have revealed an H1-dependent ATP-independent mechanism of Top II-mediatedchromatin compaction involving clamping of DNA strands[273] Thus it appears that Top II DSB handling is importantin highly diverse manipulations of chromatin packing andfurther studies are needed to elucidate the precise regulationthereof

35 Mitochondrial DNA Maintenance and Ageing Mito-chondrial DNA (mtDNA) in humans is typically a closedcircle of dsDNA [274] As in genomic DNA replication and

transcription processes exert topological stresses on mtDNAmolecules which are relieved by native topoisomerases [197275] Mammalian mitochondria contain 3 topoisomerasesencoded in the nucleus Top1mt (TOP1MT) and Top III120572(TOP3A) of the type I family and Top IIb (TOP2B) fromthe type II family of enzymes [275] Although mitochon-drial Top IIb is C-terminally truncated compared to itsnuclear counterpart it still retains basic type II topoisomeraseDSB turnover activity albeit with reduced processivity andexhibits a similar pharmacological profile [276] Top1mtdeletion inmice results in elevatedmtDNA transcription andinduction of a stress response accompanied by considerableupregulation of Top IIb suggesting a potential compensatoryrole of the type II enzyme [277] However both TOP2Bminusminus

MEFs and TOP1MTminusminusTOP2Bminusminus display wild-type levels oftranscription and only the double-knockout elicits a stressresponse suggesting nonoverlapping functions of the twomolecules [277] This picture is further complicated by therecent discovery of active full-length Top IIa and Top IIb inboth murine and human mitochondria which although asyet uncharacterised are likely involved in classical functionsin replication and transcription like their nuclear counter-parts [278]

Top IIIa is a type IA topoisomerase which is found in boththe nucleus and mitochondria and has been shown to play acrucial role in the maintenance of mtDNA genomic integrityin Drosophila Indeed loss of mitochondrial import of TopIIIa in Drosophila is associated with a reduction in mtDNAcopy number mitochondrial dysfunction impaired fertilityand accelerated ageing [279 280] The interplay betweenthese type I and II topoisomerases in the maintenance ofmtDNA and the precise roles of DSB handling therein willrequire further investigation

A recently proposed but unexplored theory suggeststhat mitochondrial Top II may be involved in ageing [275]With age mtDNA undergoes attrition through entanglementandor recombination such that mtDNA deletion is increas-ingly observed over time These deletion events are foundto contribute to dysfunction of the respiratory chain [281]and other tissue impairments associated with ageing [282ndash287] One hypothesis postulates that age-related attritionoccurs as a result of mitochondrial ldquopoisoningrdquo by exoge-nous agents resulting in dysregulation of topoisomerase II-mediated DSB handling [275] Many such ldquopoisonsrdquo disruptthe ligation step of Top II resulting in accumulation of DSB-enzyme intermediates [275] Mammalian mitochondrial TopIIb has been found to be inhibited by the anticancer drugsamsacrine and teniposide [288] and it is possible thatvarious known nuclear Top II poisons ranging from thefungal toxin alternariol [289] and chemotherapeutics [290]to dietary components such as bioflavonoids from fruit andvegetables [291] may also inhibit the truncated or full-length type II topoisomerases inmitochondria Furthermorebase oxidations abasic sites and other exogenously inducedbase modifications can act via an unknown mechanismto abnormally enhance Top II DSB formation [292] andpromote accumulation of DNA-enzyme intermediates at sitesof damage [293] Combined with the finding that DSBs in


the mitochondrial genome can induce considerable mtDNAdeletion [294] Sobek and Boege speculate that exposureto mitochondrial topoisomerase ldquopoisonsrdquo over time withage could lead to potential DSB-dependent mtDNA attrition[275] While it is likely that mitochondrial Top II performssimilar DSB-related functions to its nuclear counterpartfurther studies will be needed to elucidate the precise natureof its role in mitochondria and its contribution if any to theageing process

Further insight into the role of Top II-induced DSBs inmitochondria may be drawn from parallels in Kinetoplastidsa group of flagellated protozoa which includes the parasiticTrypanosoma and Leishmania species which possess a char-acteristic dense granule of DNA called a kinetoplast withintheir single mitochondrion The kinetoplast consists of anenormous network of catenated circular mtDNA (kDNA)comprising short minicircles and much larger maxicirclesand thus represents a considerable topological challengein replication and segregation [295] The mitochondrioncontains a type II topoisomerase which localises to thekDNA [296 297] and has been shown to be important forthe maintenance of structural integrity in the kinetoplast[298] mending of holes in the kDNA following individualminicircle release for replication [299] segregation of daugh-ter minicircles [300] and their reattachment to the kDNAnetwork [301]

4 Concluding Remarks and Future Prospects

DSB physiology is thus at its very core a molecular surgeryreliant on both calculated incisions and timely suturingthereof all the while under the intense scrutiny of numerousregulatory mechanisms While the basic biochemical mech-anisms of the processes discussed above are well establishedtheir multilateral spatiotemporal regulation remains a field ofactive research

The roles of recombination defects in human diseaseand tumorigenesis are being increasingly appreciated andfurther studies will be needed to examine the nature of thedysregulation of DSB physiology in pathogenesis New workmay reveal novel pharmacological targets for human diseasesuch as the possibility of targeting DSB signalling and repairproteins in neurodegenerative conditions such as Alzheimerrsquosdisease to lessen aberrant DSB formation

Novel tissue-specific roles of topoisomerases in neuro-physiology are only just being uncovered with future studiesrequired to elucidate the function of DSBs therein to under-stand mechanisms of learning memory and cognition Inaddition future work is necessary to characterise the natureof mitochondrial DSB physiology and the as yet unexploredimplications on mitochondrial metabolism disease andageing

Furthermore both topoisomerases and cancer DSBrepair deficiencies are important areas of investigation in thedevelopment of anticancer therapeutics and an understand-ing of the physiological roles of these targets in multiple dis-parate processes will inform the design of specific inhibitorswith fewer off-target effects

This account illustrates the breadth of DSB physiologyacross cellular biology and adds a new dimension of com-plexity to the regulation of genomic transactions on a basicbiochemical level The emerging role of DSB-dependentchanges in topology not only in replication and basaltranscription but also in activity-induced transcriptionalchanges and potential roles in the chromatin landscape andmitochondria paint a picture of a far more active role of DSBsin fundamental DNA metabolism than previously thought

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors are very grateful to Dr Joseph Maman for hisguidance and support during the production of this work andfor his ever helpful advice whenever needed

References

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[2] S P Jackson and J Bartek ldquoThe DNA-damage response inhuman biology and diseaserdquoNature vol 461 no 7267 pp 1071ndash1078 2009

[3] A Schipler and G Iliakis ldquoDNA double-strand-break com-plexity levels and their possible contributions to the probabilityfor error-prone processing and repair pathway choicerdquo NucleicAcids Research vol 41 no 16 pp 7589ndash7605 2013

[4] P A Jeggo and M Lobrich ldquoDNA double-strand breaks theircellular and clinical impactrdquoOncogene vol 26 no 56 pp 7717ndash7719 2007

[5] I Brandsma and D C Gent ldquoPathway choice in DNA doublestrand break repair observations of a balancing actrdquo GenomeIntegrity vol 3 article 9 2012

[6] D B Roth ldquoV(D)J Recombination Mechanism Errors andFidelityrdquoMicrobiology Spectrum vol 2 no 6 2014

[7] Z XuH Zan E J Pone TMai andPCasali ldquoImmunoglobulinclass-switch DNA recombination induction targeting andbeyondrdquoNature Reviews Immunology vol 12 no 7 pp 517ndash5312012

[8] I Lam and S Keeney ldquoMechanism and regulation of meioticrecombination initiationrdquo Cold Spring Harbor Perspectives inBiology vol 7 no 1 Article ID a016634 2015

[9] I Lucas T Germe M Chevrier-Miller and O HyrienldquoTopoisomerase II can unlink replicating DNA by precatenaneremovalrdquo EMBO Journal vol 20 no 22 pp 6509ndash6519 2001

[10] J Baxter and J F X Diffley ldquoTopoisomerase II InactivationPrevents theCompletion ofDNAReplication inBuddingYeastrdquoMolecular Cell vol 30 no 6 pp 790ndash802 2008

[11] S L French M L Sikes R D Hontz et al ldquoDistinguishingthe roles of topoisomerases I and II in relief of transcription-induced torsional stress in yeast rRNA genesrdquo Molecular andCellular Biology vol 31 no 3 pp 482ndash494 2011

[12] R Madabhushi F Gao and A R Pfenning ldquoActivity-inducedDNA breaks govern the expression of neuronal early-responsegenesrdquo Cell vol 161 no 7 pp 1592ndash1605 2015


[13] V K Tiwari L Burger V Nikoletopoulou et al ldquoTarget genesof Topoisomerase II120573 regulate neuronal survival and are definedby their chromatin staterdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 109 no 16 ppE934ndashE943 2012

[14] Y L Lyu C-P Lin A M Azarova L Cai J C Wangand L F Liu ldquoRole of topoisomerase II120573 in the expressionof developmentally regulated genesrdquo Molecular and CellularBiology vol 26 no 21 pp 7929ndash7941 2006

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[18] J E Deweese and N Osheroff ldquoThe DNA cleavage reactionof topoisomerase II Wolf in sheeprsquos clothingrdquo Nucleic AcidsResearch vol 37 no 3 pp 738ndash748 2009

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[24] P C Swanson and S Desiderio ldquoV(D)J recombination signalrecognition Distinct overlapping DNA- protein contacts incomplexes containing RAG1 with and without RAG2rdquo Immu-nity vol 9 no 1 pp 115ndash125 1998

[25] K Hiom and M Gellert ldquoA stable RAG1-RAG2-DNA complexthat is active in V(D)J cleavagerdquo Cell vol 88 no 1 pp 65ndash721997

[26] XMo T Bailin andM J Sadofsky ldquoRAG1 andRAG2 cooperatein specific binding to the recombination signal sequence invitrordquo The Journal of Biological Chemistry vol 274 no 11 pp7025ndash7031 1999

[27] Q M Eastman T M J Leu and D G Schatz ldquoInitiation ofV(D)J recombination in vitro obeying the 1223 rulerdquo Naturevol 380 no 6569 pp 85ndash88 1996

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[29] P C Swanson ldquoFine structure and activity of discrete RAG-HMG complexes on V(D)J recombination signalsrdquo Molecularand Cellular Biology vol 22 no 5 pp 1340ndash1351 2002

[30] D J Sawchuk FWeis-Garcia SMalik et al ldquoV(D)J recombina-tion Modulation of RAG1 and RAG2 cleavage activity on 1223substrates by whole cell extract and DNA-bending proteinsrdquoThe Journal of Experimental Medicine vol 185 no 11 pp 2025ndash2032 1997

[31] V Aidinis T Bonaldi M Beltrame S Santagata M E Bianchiand E Spanopoulou ldquoThe RAG1 homeodomain recruits HMG1and HMG2 to facilitate recombination signal sequence bindingand to enhance the intrinsic DNA-bending activity of RAG1-RAG2rdquoMolecular and Cellular Biology vol 19 no 10 pp 6532ndash6542 1999

[32] K K Rodgers I J Villey L Ptaszek E Corbett D G Schatzand J E Coleman ldquoA dimer of the lymphoid protein RAG1recognizes the recombination signal sequence and the complexstably incorporates the high mobility group protein HMG2rdquoNucleic Acids Research vol 27 no 14 pp 2938ndash2946 1999

[33] D C Van Gent K Hiom T T Paull and M Gellert ldquoStimula-tion of V(D)J cleavage by high mobility group proteinsrdquo EMBOJournal vol 16 no 10 pp 2665ndash2670 1997

[34] M J Difilippantonio C J McMahan Q M Eastman E Spa-nopoulou and D G Schatz ldquoRAG1 mediates signal sequencerecognition and recruitment of RAG2 inV(D)J recombinationrdquoCell vol 87 no 2 pp 253ndash262 1996

[35] Y Akamatsu and M A Oettinger ldquoDistinct roles of RAG1 andRAG2 in binding the V(D)J recombination signal sequencesrdquoMolecular and Cellular Biology vol 18 no 8 pp 4670ndash46781998

[36] D C Van Gent K Mizuuchi and M Gellert ldquoSimilaritiesbetween initiation of V(D)J recombination and retroviral inte-grationrdquo Science vol 271 no 5255 pp 1592ndash1594 1996

[37] A Agrawal and D G Schatz ldquoRAG1 and RAG2 form a stablepostcleavage synaptic complex with DNA containing signalends inV(D)J recombinationrdquoCell vol 89 no 1 pp 43ndash53 1997

[38] Y Ma U Pannicke K Schwarz and M R Lieber ldquoHair-pin opening and overhang processing by an ArtemisDNA-dependent protein kinase complex in nonhomologous endjoining and V(D)J recombinationrdquo Cell vol 108 no 6 pp 781ndash794 2002

[39] M S Schlissel ldquoDoes Artemis end the hunt for the hairpin-opening activity in V(D)J recombinationrdquo Cell vol 109 no 1pp 1ndash4 2002

[40] E Besmer J Mansilla-Soto S Cassard et al ldquoHairpin codingend opening is mediated by RAG1 and RAG2 proteinsrdquoMolec-ular Cell vol 2 no 6 pp 817ndash828 1998

[41] P E Shockett and D G Schatz ldquoDNA hairpin openingmediated by the RAG21 and RAG2 proteinsrdquo Molecular andCellular Biology vol 19 no 6 pp 4159ndash4166 1999

[42] S Santagata E Besmer A Villa et al ldquoThe RAG1RAG2complex constitutes a 3rsquo flap endonuclease Implications forjunctional diversity in V(D)J and transpositional recombina-tionrdquoMolecular Cell vol 4 no 6 pp 935ndash947 1999

[43] C-L Tsai A H Drejer and D G Schatz ldquoEvidence of acritical architectural function for the RAG proteins in endprocessing protection and joining in V(D)J recombinationrdquoGenes amp Development vol 16 no 15 pp 1934ndash1949 2002

[44] S M Lewis ldquoThe Mechanism of V(D)J Joining Lessonsfrom Molecular Immunological and Comparative AnalysesrdquoAdvances in Immunology vol 56 no C pp 27ndash150 1994

[45] G H Gauss and M R Lieber ldquoMechanistic constraints ondiversity in human V(D)J recombinationrdquo Molecular and Cel-lular Biology vol 16 no 1 pp 258ndash269 1996


[46] S E Critchlow R P Bowater and S P Jackson ldquoMammalianDNA double-strand break repair protein XRCC4 interacts withDNA ligase IVrdquo Current Biology vol 7 no 8 pp 588ndash598 1997

[47] U Grawunder M Wilm X Wu et al ldquoActivity of DNA ligaseIV stimulated by complex formation with XRCC4 protein inmammalian cellsrdquo Nature vol 388 no 6641 pp 492ndash495 1997

[48] S Costantini L Woodbine L Andreoli P A Jeggo and AVindigni ldquoInteraction of the Ku heterodimer with the DNAligase IVXrcc4 complex and its regulation by DNA-PKrdquo DNARepair vol 6 no 6 pp 712ndash722 2007

[49] S Mickelsen C Snyder K Trujillo M Bogue D B Roth andK Meek ldquoModulation of terminal deoxynucleotidyltransferaseactivity by the DNA- dependent protein kinaserdquoThe Journal ofImmunology vol 163 no 2 pp 834ndash843 1999

[50] S A N McElhinny J M Havener M Garcia-Diaz et alldquoA gradient of template dependence defines distinct biologicalroles for family X polymerases in nonhomologous end joiningrdquoMolecular Cell vol 19 no 3 pp 357ndash366 2005

[51] B Bertocci A De Smet J-C Weill and C-A Reynaud ldquoNon-overlapping Functions of DNA Polymerases Mu Lambda andTerminal Deoxynucleotidyltransferase during Immunoglobu-lin V(D)J Recombination In Vivordquo Immunity vol 25 no 1 pp31ndash41 2006

[52] P Ahnesorg P Smith and S P Jackson ldquoXLF interacts with theXRCC4-DNA Ligase IV complex to promote DNA nonhomol-ogous end-joiningrdquo Cell vol 124 no 2 pp 301ndash313 2006

[53] Z Li D I Dordai J Lee and S Desiderio ldquoA conserveddegradation signal regulates RAG-2 accumulation during celldivision and links V(D)J recombination to the cell cyclerdquoImmunity vol 5 no 6 pp 575ndash589 1996

[54] W C Lin and S Desiderio ldquoCell cycle regulation of V(D)Jrecombination-activating protein RAG-2rdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 91 no 7 pp 2733ndash2737 1994

[55] L Zhang T L Reynolds X Shan and S Desiderio ldquoCouplingof V(D)J Recombination to the Cell Cycle Suppresses GenomicInstability and Lymphoid Tumorigenesisrdquo Immunity vol 34no 2 pp 163ndash174 2011

[56] P Stanhope-Baker K M Hudson A L Shaffer A Constanti-nescu and M S Schlissel ldquoCell type-specific chromatin struc-ture determines the targeting of V(D)J recombinase activity invitrordquo Cell vol 85 no 6 pp 887ndash897 1996

[57] G D Yancopoulos and F W Alt ldquoDevelopmentally controlledand tissue-specific expression of unrearranged VH gene seg-mentsrdquo Cell vol 40 no 2 pp 271ndash281 1985

[58] S M Lewis E Agard S Suh and L Czyzyk ldquoCryptic signalsand the fidelity of V(D)J joiningrdquo Molecular and CellularBiology vol 17 no 6 pp 3125ndash3136 1997

[59] B P Sleckman J RGorman andFWAlt ldquoAccessibility controlof antigen-receptor variable-region gene assembly Role of cis-acting elementsrdquo Annual Review of Immunology vol 14 pp459ndash481 1996

[60] F McBlane and J Boyes ldquoStimulation of V(D)J recombinationby histone acetylationrdquo Current Biology vol 10 no 8 pp 483ndash486 2000

[61] J Kwon A N Imbalzano A Matthews and M A OettingerldquoAccessibility of nucleosomal DNA to V(D)J cleavage is modu-lated by RSS positioning and HMG1rdquoMolecular Cell vol 2 no6 pp 829ndash839 1998

[62] A Golding S Chandler E Ballestar A P Wolffe and M SSchlissel ldquoNucleosome structure completely inhibits in vitro

cleavage by the V(D)J recombinaserdquo EMBO Journal vol 18 no13 pp 3712ndash3723 1999

[63] D B Roth and S Y Roth ldquoUnequal access Regulating V(D)Jrecombination through chromatin remodelingrdquo Cell vol 103no 5 pp 699ndash702 2000

[64] D G Schatz and Y Ji ldquoRecombination centres and the orches-tration of V(D)J recombinationrdquo Nature Reviews Immunologyvol 11 no 4 pp 251ndash263 2011

[65] A G W Matthews A J Kuo S Ramon-Maiques et al ldquoRAG2PHD finger couples histone H3 lysine 4 trimethylation withV(D)J recombinationrdquo Nature vol 450 no 7172 pp 1106ndash11102007

[66] N Shimazaki A G Tsai and M R Lieber ldquoH3K4me3 Stimu-lates the V(D)J RAG Complex for Both Nicking and Hairpin-ning in trans in Addition to Tethering in cis Implications forTranslocationsrdquoMolecular Cell vol 34 no 5 pp 535ndash544 2009

[67] P Mombaerts J Iacomini R S Johnson K Herrup S Tone-gawa and V E Papaioannou ldquoRAG-1-deficient mice have nomature B and T lymphocytesrdquo Cell vol 68 no 5 pp 869ndash8771992

[68] Y Shinkai K-P Lam E M Oltz V Stewart M Mendelsohn JCharron et al ldquoRAG-2-deficientmice lackmature lymphocytesowing to inability to initiate V(D)J rearrangementrdquoCell vol 68no 5 pp 855ndash867 1992

[69] A Villa S Santagata F Bozzi et al ldquoPartial V(D)J recombina-tion activity leads to omenn syndromerdquo Cell vol 93 no 5 pp885ndash896 1998

[70] J Wang J M Pluth P K Cooper M J Cowan D J Chenand S M Yannone ldquoArtemis deficiency confers a DNA double-strand break repair defect and Artemis phosphorylation statusis altered by DNA damage and cell cycle progressionrdquo DNARepair vol 4 no 5 pp 556ndash570 2005

[71] Y Gao J Chaudhuri C Zhu L Davidson D T Weaver andF W Alt ldquoA targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombinationrdquoImmunity vol 9 no 3 pp 367ndash376 1998

[72] S Rooney J Sekiguchi C Zhu et al ldquoLeaky Scid PhenotypeAssociated with Defective V(D)J Coding End Processing inArtemis-DeficientMicerdquoMolecular Cell vol 10 no 6 pp 1379ndash1390 2002

[73] M Ege Y Ma B Manfras et al ldquoOmenn syndrome due toARTEMIS mutationsrdquo Blood vol 105 no 11 pp 4179ndash41862005

[74] A Nussenzweig K Sokol P Burgman L Li and G C LildquoHypersensitivity of Ku80-deficient cell lines and mice to DNAdamage The effects of ionizing radiation on growth survivaland developmentrdquo Proceedings of the National Acadamy ofSciences of the United States of America vol 94 no 25 pp13588ndash13593 1997

[75] Y Gu K J Seidl G A Rathbun et al ldquoGrowth retardation andleaky SCID phenotype of Ku70-deficient micerdquo Immunity vol7 no 5 pp 653ndash665 1997

[76] S Jin and D T Weaver ldquoDouble-strand break repair byKu70 requires heterodimerization with Ku80 andDNA bindingfunctionsrdquo EMBO Journal vol 16 no 22 pp 6874ndash6885 1997

[77] Y Gao DO FergusonW Xie et al ldquoInterplay of p53 andDNA-repair protein XRCC4 in tumorigenesis genomic stability anddevelopmentrdquo Nature vol 404 no 6780 pp 897ndash900 2000

[78] K M Frank N E Sharpless Y Gao et al ldquoDNA ligaseIV deficiency in mice leads to defective neurogenesis andembryonic lethality via the p53 pathwayrdquoMolecular Cell vol 5no 6 pp 993ndash1002 2000


[79] Y Gao Y Sun K M Frank et al ldquoA critical role for DNA end-joining proteins in both lymphogenesis and neurogenesisrdquoCellvol 95 no 7 pp 891ndash902 1998

[80] R Taub I Kirsch C Morton et al ldquoTranslocation of thec-myc gene into the immunoglobulin heavy chain locus inhuman Burkitt lymphoma and murine plasmacytoma cellsrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 79 no 24 I pp 7837ndash7841 1982

[81] OMonni R Oinonen E Elonen et al ldquoGain of 3q and deletionof 11q22 are frequent aberrations in mantle cell lymphomardquoGenes Chromosomes and Cancer vol 21 no 4 pp 298ndash3071998

[82] R Baer K-C Chen S D Smith and T H Rabbitts ldquoFusion ofan immunoglobulin variable gene and a T cell receptor constantgene in the chromosome 14 inversion associated with T celltumorsrdquo Cell vol 43 no 3 pp 705ndash713 1985

[83] R J Bende L A Smit and C J M van Noesel ldquoMolecularpathways in follicular lymphomardquo Leukemia vol 21 no 1 pp18ndash29 2007

[84] S C Raghavan P C Swanson X Wu C-L Hsieh and M RLieber ldquoA non-B-DNA structure at the Bcl-2 major breakpointregion is cleaved by the RAG complexrdquo Nature vol 428 no6978 pp 88ndash93 2004

[85] X Cui Z Lu A Kurosawa et al ldquoBoth CpG methylation andactivation-induced deaminase are required for the fragility ofthe human bcl-2 major breakpoint region Implications for thetiming of the breaks in the t(1418) translocationrdquoMolecular andCellular Biology vol 33 no 5 pp 947ndash957 2013

[86] D B Roth ldquoRestraining the V(D)J recombinaserdquo NatureReviews Immunology vol 3 no 8 pp 656ndash666 2003

[87] J Chaudhuri and F W Alt ldquoClass-switch recombinationInterplay of transcription DNA deamination and DNA repairrdquoNature Reviews Immunology vol 4 no 7 pp 541ndash552 2004

[88] W Dunnick G Z Hertz L Scappino and C GritzmacherldquoDNA sequences at immunoglobulin switch region recombina-tion sitesrdquo Nucleic Acids Research vol 21 no 3 pp 365ndash3721993

[89] J S Rush S D Fugmann and D G Schatz ldquoStaggered AID-dependent DNA double strand breaks are the predominantDNA lesions targeted to S120583 in Ig class switch recombinationrdquoInternational Immunology vol 16 no 4 pp 549ndash557 2004

[90] N Catalan F Selz K Imai P Revy A Fischer and A DurandyldquoThe block in immunoglobulin class switch recombinationcaused by activation-induced cytidine deaminase deficiencyoccurs prior to the generation of DNA double strand breaks inswitch 120583 regionrdquoThe Journal of Immunology vol 171 no 5 pp2504ndash2509 2003

[91] M Muramatsu K Kinoshita S Fagarasan S Yamada YShinkai and T Honjo ldquoClass switch recombination andhypermutation require activation-induced cytidine deaminase(AID) a potential RNA editing enzymerdquo Cell vol 102 no 5 pp553ndash563 2000

[92] S K Petersen-Mahrt R S Harris and M S Neuberger ldquoAIDmutates E coli suggesting a DNA deamination mechanism forantibody diversificationrdquo Nature vol 418 no 6893 pp 99ndash1032002

[93] C E Schrader E K Linehan S NMochegova R TWoodlandand J Stavnezer ldquoInducible DNA breaks in Ig S regions aredependent on AID and UNGrdquo The Journal of ExperimentalMedicine vol 202 no 4 pp 561ndash568 2005

[94] C Rada G T Williams H Nilsen D E Barnes T Lindahland M S Neuberger ldquoImmunoglobulin isotype switching

is inhibited and somatic hypermutation perturbed in UNG-deficient micerdquo Current Biology vol 12 no 20 pp 1748ndash17552002

[95] J E J Guikema E K Linehan D Tsuchimoto et al ldquoAPE1- AndAPE2-dependent DNA breaks in immunoglobulin class switchrecombinationrdquoThe Journal of Experimental Medicine vol 204no 12 pp 3017ndash3026 2007

[96] J Stavnezer andC E Schrader ldquoMismatch repair converts AID-instigated nicks to double-strand breaks for antibody class-switch recombinationrdquo Trends in Genetics vol 22 no 1 pp 23ndash28 2006

[97] G Bastianello andHArakawa ldquoAdouble-strand break can trig-ger immunoglobulin gene conversionrdquo Nucleic Acids Researchvol 45 no 1 pp 231ndash243 2017

[98] C Rada J M Di Noia and M S Neuberger ldquoMismatchrecognition and uracil excision provide complementary pathsto both Ig switching and the AT-focused phase of somaticmutationrdquoMolecular Cell vol 16 no 2 pp 163ndash171 2004

[99] M R Ehrenstein and M S Neuberger ldquoDeficiency inMsh2 affects the efficiency and local sequence specificity ofimmunoglobulin class-switch recombination Parallels withsomatic hypermutationrdquo EMBO Journal vol 18 no 12 pp3484ndash3490 1999

[100] A Martin Z Li D P Lin et al ldquoMsh2 ATPase Activity IsEssential for Somatic Hypermutation at A-T Basepairs and forEfficient Class Switch Recombinationrdquo The Journal of Experi-mental Medicine vol 198 no 8 pp 1171ndash1178 2003

[101] S A Martomo W W Yang and P J Gearhart ldquoA role forMsh6 but not Msh3 in somatic hypermutation and class switchrecombinationrdquoThe Journal of Experimental Medicine vol 200no 1 pp 61ndash68 2004

[102] C E Schrader J Vardo and J Stavnezer ldquoRole for mismatchrepair proteins Msh2 Mlh1 and Pms2 in immunoglobulinclass switching shown by sequence analysis of recombinationjunctionsrdquoThe Journal of Experimental Medicine vol 195 no 3pp 367ndash373 2002

[103] H M Shen A Tanaka G Bozek D Nicolae and UStorb ldquoSomatic hypermutation and class switch recombinationin Msh6 --Ung-- double-knockout micerdquo The Journal ofImmunology vol 177 no 8 pp 5386ndash5392 2006

[104] P D Bardwell C J Woo K Wei et al ldquoAltered somatichypermutation and reduced class-switch recombination inexonuclease 1-mutant micerdquo Nature Immunology vol 5 no 2pp 224ndash229 2004

[105] J Stavnezer J E J Guikema and C E Schrader ldquoMechanismand regulation of class switch recombinationrdquoAnnual Review ofImmunology vol 26 pp 261ndash292 2008

[106] A JMatthews S Zheng L J DiMenna and J Chaudhuri ldquoReg-ulation of immunoglobulin class-switch recombination Chore-ography of noncoding transcription targeted DNA deamina-tion and long-rangeDNA repairrdquoAdvances in Immunology vol122 pp 1ndash57 2014

[107] R Casellas ANussenzweig RWuerffel et al ldquoKu80 is requiredfor immunoglobulin isotype switchingrdquo EMBO Journal vol 17no 8 pp 2404ndash2411 1998

[108] J P Manis Y Gu R Lansford et al ldquoKu70 is required forlate B cell development and immunoglobulin heavy chain classswitchingrdquo The Journal of Experimental Medicine vol 187 no12 pp 2081ndash2089 1998

[109] P Soulas-Sprauel G Le Guyader P Rivera-Munoz et al ldquoRolefor DNA repair factor XRCC4 in immunoglobulin class switch


recombinationrdquoThe Journal of Experimental Medicine vol 204no 7 pp 1717ndash1727 2007

[110] L Han and K Yu ldquoAltered kinetics of nonhomologous endjoining and class switch recombination in ligase IV-deficient BcellsrdquoThe Journal of Experimental Medicine vol 205 no 12 pp2745ndash2753 2008

[111] A Bottaro R Lansford L Xu J Zhang P Rothman andF W Alt ldquoS region transcription per se promotes basal IgEclass switch recombination but additional factors regulate theefficiency of the processrdquo EMBO Journal vol 13 no 3 pp 665ndash674 1994

[112] S Jung K Rajewsky and A Radbruch ldquoShutdown of classswitch recombination by deletion of a switch region controlelementrdquo Science vol 259 no 5097 pp 984ndash987 1993

[113] K Kinoshita J Tashiro S Tomita C-G Lee and T HonjoldquoTarget specificity of immunoglobulin class switch recombina-tion is not determined by nucleotide sequences of S regionsrdquoImmunity vol 9 no 6 pp 849ndash858 1998

[114] Y Nambu M Sugai H Gonda et al ldquoTranscription-CoupledEvents Associating with Immunoglobulin Switch Region Chro-matinrdquo Science vol 302 no 5653 pp 2137ndash2140 2003

[115] J Chaudhuri M Tian C Khuong K Chua E Pinaud and FW Alt ldquoTranscription-targeted DNA deamination by the AIDantibody diversification enzymerdquoNature vol 422 no 6933 pp726ndash730 2003

[116] D Ronai M D Iglesias-Ussel M Fan Z Li A Martinand M D Scharff ldquoDetection of chromatin-associated single-stranded DNA in regions targeted for somatic hypermutationrdquoThe Journal of Experimental Medicine vol 204 no 1 pp 181ndash190 2007

[117] F-T Huang K Yu B B Balter et al ldquoSequence dependence ofchromosomal R-loops at the immunoglobulin heavy-chain S120583class switch regionrdquoMolecular and Cellular Biology vol 27 no16 pp 5921ndash5932 2007

[118] K Yu F Chedin C-L Hsieh T E Wilson and M RLieber ldquoR-loops at immunoglobulin class switch regions in thechromosomes of stimulated B cellsrdquoNature Immunology vol 4no 5 pp 442ndash451 2003

[119] R Pavri A Gazumyan M Jankovic et al ldquoActivation-inducedcytidine deaminase targets DNA at sites of RNA polymerase IIstalling by interactionwith Spt5rdquoCell vol 143 no 1 pp 122ndash1332010

[120] L Wang R Wuerffel S Feldman A A Khamlichi and A LKenter ldquoS region sequence RNA polymerase II and histonemodifications create chromatin accessibility during class switchrecombinationrdquoThe Journal of Experimental Medicine vol 206no 8 pp 1817ndash1830 2009

[121] L Wang N Whang R Wuerffel and A L Kenter ldquoAID-dependent histone acetylation is detected in immunoglobulinS regionsrdquoThe Journal of Experimental Medicine vol 203 no 1pp 215ndash226 2006

[122] H Ohkura ldquoMeiosis An overview of key differences frommitosisrdquo Cold Spring Harbor Perspectives in Biology vol 7 no5 pp 1ndash15 2015

[123] D K Bishop and D Zickler ldquoEarly decision Meiotic crossoverinterference prior to stable strand exchange and synapsisrdquo Cellvol 117 no 1 pp 9ndash15 2004

[124] S Keeney C N Giroux and N Kleckner ldquoMeiosis-specificDNA double-strand breaks are catalyzed by Spo11 a memberof a widely conserved protein familyrdquo Cell vol 88 no 3 pp375ndash384 1997

[125] J Liu T-C Wu and M Lichten ldquoThe location and structureof double-strand DNA breaks induced during yeast meiosisEvidence for a covalently linked DNA-protein intermediaterdquoEMBO Journal vol 14 no 18 pp 4599ndash4608 1995

[126] S Keeney and M J Neale ldquoInitiation of meiotic recombinationby formation of DNA double-strand breaks Mechanism andregulationrdquo Biochemical Society Transactions vol 34 no 4 pp523ndash525 2006

[127] A L Marston and A Amon ldquoMeiosis Cell-cycle controlsshuffle and dealrdquo Nature Reviews Molecular Cell Biology vol 5no 12 pp 983ndash997 2004

[128] P J Romanienko and R D Camerini-Otero ldquoCloning char-acterization and localization of mouse and human SPO11rdquoGenomics vol 61 no 2 pp 156ndash169 1999

[129] P J Romanienko and R D Camerini-Otero ldquoThe mouse Spo11gene is required for meiotic chromosome synapsisrdquo MolecularCell vol 6 no 5 pp 975ndash987 2000

[130] C Arora K Kee S Maleki and S Keeney ldquoAntiviral proteinSki8 is a direct partner of Spo11 in meiotic DNA break forma-tion independent of its cytoplasmic role in RNA metabolismrdquoMolecular Cell vol 13 no 4 pp 549ndash559 2004

[131] K Kee R U Protacio C Arora and S Keeney ldquoSpatialorganization and dynamics of the association of Rec102 andRec104 with meiotic chromosomesrdquo EMBO Journal vol 23 no8 pp 1815ndash1824 2004

[132] K Kee and S Keeney ldquoFunctional interactions between SPO11and REC102 during initiation of meiotic recombination inSaccharomyces cerevisiaerdquo Genetics vol 160 no 1 pp 111ndash1222002

[133] H Sasanuma H Murakami T Fukuda T Shibata A Nicolasand K Ohta ldquoMeiotic association between Spo11 regulated byRec102 Rec104 and Rec114rdquo Nucleic Acids Research vol 35 no4 pp 1119ndash1133 2007

[134] J Li GWHooker andG S Roeder ldquoSaccharomyces cerevisiaeMer2 Mei4 and Rec114 form a complex required for meioticdouble-strand break formationrdquo Genetics vol 173 no 4 pp1969ndash1981 2006

[135] M J Neale J Pan and S Keeney ldquoEndonucleolytic processingof covalent protein-linked DNA double-strand breaksrdquo Naturevol 436 no 7053 pp 1053ndash1057 2005

[136] T Usui T Ohta H Oshiumi J-I Tomizawa H Ogawa and TOgawa ldquoComplex formation and functional versatility of Mre11of budding yeast in recombinationrdquo Cell vol 95 no 5 pp 705ndash716 1998

[137] J E Haber ldquoThe many interfaces of Mre11rdquo Cell vol 95 no 5pp 583ndash586 1998

[138] N Manfrini I Guerini A Citterio G Lucchini and M PLonghese ldquoProcessing of meiotic DNA double strand breaksrequires cyclin-dependent kinase and multiple nucleasesrdquo TheJournal of Biological Chemistry vol 285 no 15 pp 11628ndash116372010

[139] D A Bressan B K Baxter and J H J Petrini ldquoTheMre11-Rad50-Xrs2 protein complex facilitates homologousrecombination-based double-strand break repair in Saccha-romyces cerevisiaerdquoMolecular and Cellular Biology vol 19 no11 pp 7681ndash7687 1999

[140] S L Andersen and J Sekelsky ldquoMeiotic versus mitotic recom-bination Two different routes for double-strand break repairThe different functions of meiotic versus mitotic DSB repair arereflected in different pathway usage and different outcomesrdquoBioEssays vol 32 no 12 pp 1058ndash1066 2010


[141] H Sun D Treco and J W Szostak ldquoExtensive 31015840-overhangingsingle-stranded DNA associated with the meiosis-specificdouble-strand breaks at the ARG4 recombination initiationsiterdquo Cell vol 64 no 6 pp 1155ndash1161 1991

[142] F Baudat Y Imai and B De Massy ldquoMeiotic recombinationin mammals Localization and regulationrdquo Nature ReviewsGenetics vol 14 no 11 pp 794ndash806 2013

[143] M J Neale and S Keeney ldquoClarifying the mechanics of DNAstrand exchange inmeiotic recombinationrdquoNature vol 442 no7099 pp 153ndash158 2006

[144] J Fraune S Schramm M Alsheimer and R BenaventeldquoThemammalian synaptonemal complex Protein componentsassembly and role in meiotic recombinationrdquo Experimental CellResearch vol 318 no 12 pp 1340ndash1346 2012

[145] N Hunter ldquoMeiotic recombination The essence of heredityrdquoCold Spring Harbor Perspectives in Biology vol 7 no 12 ArticleID a016618 2015

[146] V Borde A S H Goldman and M Lichten ldquoDirect couplingbetween meiotic DNA replication and recombination initia-tionrdquo Science vol 290 no 5492 pp 806ndash809 2000

[147] R Padmore L Cao and N Kleckner ldquoTemporal comparisonof recombination and synaptonemal complex formation duringmeiosis in S cerevisiaerdquo Cell vol 66 no 6 pp 1239ndash1256 1991

[148] S K Mahadevaiah J M A Turner F Baudat et al ldquoRecombi-national DNA double-strand breaks in mice precede synapsisrdquoNature Genetics vol 27 no 3 pp 271ndash276 2001

[149] J K Jang D E Sherizen R Bhagat E A Manheim and K SMcKim ldquoRelationship ofDNAdouble-strand breaks to synapsisin Drosophilardquo Journal of Cell Science vol 116 no 15 pp 3069ndash3077 2003

[150] HMurakami and S Keeney ldquoRegulating the formation of DNAdouble-strand breaks inmeiosisrdquoGenes ampDevelopment vol 22no 3 pp 286ndash292 2008

[151] T Odorisio T A Rodriguez E P Evans A R Clarke and P SBurgoyne ldquoThe meiotic checkpoint monitoring synapsis elim-inates spermatocytes via p53-independent apoptosisrdquo NatureGenetics vol 18 no 3 pp 257ndash261 1998

[152] D Lydall Y Nikolsky D K Bishop and T Weinert ldquoA meioticrecombination checkpoint controlled by mitotic checkpointgenesrdquo Nature vol 383 no 6603 pp 841ndash844 1996

[153] G S Roeder and J M Bailis ldquoThe pachytene checkpointrdquoTrends in Genetics vol 16 no 9 pp 395ndash403 2000

[154] A Hochwagen and A Amon ldquoChecking your breaks Surveil-lance mechanisms of meiotic recombinationrdquo Current Biologyvol 16 no 6 pp R217ndashR228 2006

[155] K A Henderson K Kee S Maleki P A Santini and S KeeneyldquoCyclin-Dependent KinaseDirectly Regulates Initiation ofMei-otic Recombinationrdquo Cell vol 125 no 7 pp 1321ndash1332 2006

[156] D Stuart and C Wittenberg ldquoCLB5 and CLB6 are required forpremeiotic DNA replication and activation of the meiotic SMcheckpointrdquo Genes amp Development vol 12 no 17 pp 2698ndash2710 1998

[157] L Wan H Niu B Futcher et al ldquoCdc28-Clb5 (CDK-S) andCdc7-Dbf4 (DDK) collaborate to initiate meiotic recombina-tion in yeastrdquo Genes amp Development vol 22 no 3 pp 386ndash3972008

[158] H Sasanuma K Hirota T Fukuda et al ldquoCdc7-dependentphosphorylation of Mer2 facilitates initiation of yeast meioticrecombinationrdquo Genes amp Development vol 22 no 3 pp 398ndash410 2008

[159] J Matos J J Lipp A Bogdanova et al ldquoDbf4-DependentCdc7 Kinase Links DNA Replication to the Segregation ofHomologous Chromosomes in Meiosis Irdquo Cell vol 135 no 4pp 662ndash678 2008

[160] H Murakami and S Keeney ldquoTemporospatial coordination ofmeiotic dna replication and recombination via DDK recruit-ment to replisomesrdquo Cell vol 158 no 4 pp 861ndash873 2014

[161] J Engebrecht K Voelkel-Meiman and G S Roeder ldquoMeiosis-specific RNA splicing in yeastrdquoCell vol 66 no 6 pp 1257ndash12681991

[162] J Engebrecht and G S Roeder ldquoMER1 a yeast gene requiredfor chromosome pairing and genetic recombination is inducedin meiosisrdquo Molecular and Cellular Biology vol 10 no 5 pp2379ndash2389 1990

[163] M Lichten and A S H Goldman ldquoMeiotic recombinationhotspotsrdquoAnnual Review of Genetics vol 29 pp 423ndash444 1995

[164] J L Gerton J DeRisi R Shroff M Lichten P O Brownand T D Petes ldquoGlobal mapping of meiotic recombinationhotspots and coldspots in the yeast Saccharomyces cerevisiaerdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 97 no 21 pp 11383ndash11390 2000

[165] M Sasaki J Lange and S Keeney ldquoGenome destabilization byhomologous recombination in the germ linerdquo Nature ReviewsMolecular Cell Biology vol 11 no 3 pp 182ndash195 2010

[166] B Rockmill K Voelkel-Meiman and G S Roeder ldquoCentro-mere-proximal crossovers are associated with precocious sep-aration of sister chromatids during meiosis in Saccharomycescerevisiaerdquo Genetics vol 174 no 4 pp 1745ndash1754 2006

[167] J Buard P Barthes C Grey and B De Massy ldquoDistincthistone modifications define initiation and repair of meioticrecombination in the mouserdquo EMBO Journal vol 28 no 17 pp2616ndash2624 2009

[168] F Smagulova I V Gregoretti K Brick P Khil R D Camerini-Otero and G V Petukhova ldquoGenome-wide analysis revealsnovel molecular features of mouse recombination hotspotsrdquoNature vol 472 no 7343 pp 375ndash378 2011

[169] F Baudat J Buard C Grey et al ldquoPRDM9 is a major determi-nant of meiotic recombination hotspots in humans and micerdquoScience vol 327 no 5967 pp 836ndash840 2010

[170] E D Parvanov P M Petkov and K Paigen ldquoPrdm9 controlsactivation of mammalian recombination hotspotsrdquo Science vol327 no 5967 p 835 2010

[171] K Hayashi K Yoshida and Y Matsui ldquoA histone H3 methyl-transferase controls epigenetic events required for meioticprophaserdquo Nature vol 438 no 7066 pp 374ndash378 2005

[172] T Billings E D Parvanov C L Baker M Walker K Paigenand P M Petkov ldquoDNA binding specificities of the long zinc-finger recombination protein PRDM9rdquoGenome Biology vol 14no 4 article R35 2013

[173] CGrey P Barthes G Friec F Langa F Baudat andB deMassyldquoMouse Prdm9 DNA-binding specificity determines sites ofhistone H3 lysine 4 trimethylation for initiation of meioticrecombinationrdquo PLoS Biology vol 9 no 10 Article ID e10011762011

[174] S Myers R Bowden A Tumian et al ldquoDrive against hotspotmotifs in primates implicates the PRDM9 gene in meioticrecombinationrdquo Science vol 327 no 5967 pp 876ndash879 2010

[175] L Kauppi M Barchi J Lange F Baudat M Jasin and SKeeney ldquoNumerical constraints and feedback control of double-strand breaks in mouse meiosisrdquo Genes amp Development vol 27no 8 pp 873ndash886 2013


[176] A Schwacha and N Kleckner ldquoInterhomolog bias duringmeiotic recombination Meiotic functions promote a highlydifferentiated interhomolog-only pathwayrdquo Cell vol 90 no 6pp 1123ndash1135 1997

[177] N Joshi M S Brown D K Bishop and G V Borner ldquoGrad-ual Implementation of the Meiotic Recombination Programvia Checkpoint Pathways Controlled by Global DSB LevelsrdquoMolecular Cell vol 57 no 5 pp 797ndash811 2015

[178] N Kleckner ldquoChiasma formation Chromatinaxis interplayand the role(s) of the synaptonemal complexrdquo Chromosomavol 115 no 3 pp 175ndash194 2006

[179] S Panizza M AMendozaM Berlinger et al ldquoSpo11-accessoryproteins link double-strand break sites to the chromosome axisin early meiotic recombinationrdquo Cell vol 146 no 3 pp 372ndash383 2011

[180] J M Berger S J Gamblin S C Harrison and J C WangldquoStructure and mechanism of DNA topoisomerase IIrdquo Naturevol 379 no 6562 pp 225ndash232 1996

[181] A J Schoeffler and J M Berger ldquoDNA topoisomerasesharnessing and constraining energy to govern chromosometopologyrdquo Quarterly Reviews of Biophysics vol 41 no 1 pp 41ndash101 2008

[182] J Roca ldquoThe mechanisms of DNA topoisomerasesrdquo Trends inBiochemical Sciences vol 20 no 4 pp 156ndash160 1995

[183] A J Lang S E L Mirski H J Cummings Q Yu J H Gerlachand S P C Cole ldquoStructural organization of the human TOP2Aand TOP2B genesrdquo Gene vol 221 no 2 pp 255ndash266 1998

[184] C A Austin and K L Marsh ldquoEukaryotic DNA topoisomeraseII120573rdquo BioEssays vol 20 no 3 pp 215ndash226 1998

[185] A P Null J Hudson and G J Gorbsky ldquoBoth alpha andbeta isoforms of mammalian DNA topoisomerase II associatewith chromosomes in mitosisrdquo Cell Growth amp DifferentiationThe Molecular Biology Journal of the American Association forCancer Research American Association for Cancer Research vol13 no 7 pp 325ndash333 2002

[186] N Chaly X Chen J Dentry and D L Brown ldquoOrganization ofDNA topoisomerase II isotypes during the cell cycle of humanlymphocytes and HeLa cellsrdquo Chromosome Research vol 4 no6 pp 457ndash466 1996

[187] E L Meczes K L Gilroy K L West and C A Austin ldquoTheimpact of the human DNA topoisomerse II C-terminal domainon activityrdquo PLoS ONE vol 3 no 3 Article ID e1754 2008

[188] R M Linka A C G Porter A Volkov C Mielke F Boege andM O Christensen ldquoC-Terminal regions of topoisomerase II120572and II120573 determine isoform-specific functioning of the enzymesin vivordquo Nucleic Acids Research vol 35 no 11 pp 3810ndash38222007

[189] H Turley M Comley S Houlbrook et al ldquoThe distributionand expression of the two isoforms of DNA topoisomerase II innormal and neoplastic human tissuesrdquoBritish Journal of Cancervol 75 no 9 pp 1340ndash1346 1997

[190] M E Bauman J AHolden KA BrownWGHarker and S LPerkins ldquoDifferential immunohistochemical staining for DNAtopoisomerase II alpha and beta in human tissues and for DNAtopoisomerase II beta in non-Hodgkinrsquos lymphomasrdquo ModernPathology An Official Journal of the United States and CanadianAcademy of Pathology vol 10 no 3 pp 168ndash175 1997

[191] B-G Ju V V Lunyak V Perissi et al ldquoA topoisomerase II120573-mediated dsDNA break required for regulated transcriptionrdquoScience vol 312 no 5781 pp 1798ndash1802 2006

[192] R J Lewis F T F Tsai and D B Wigley ldquoMolecular mecha-nisms of drug inhibition of DNA gyraserdquo BioEssays vol 18 no8 pp 661ndash671 1996

[193] Y-C Tse-Dinh ldquoExploring DNA topoisomerases as targets ofnovel therapeutic agents in the treatment of infectious diseasesrdquoInfectious Disorders - Drug Targets vol 7 no 1 pp 3ndash9 2007

[194] S Salerno F Da Settimo S Taliani et al ldquoRecent advances inthe development of dual topoisomerase I and II inhibitors asanticancer drugsrdquo Current Medicinal Chemistry vol 17 no 35pp 4270ndash4290 2010

[195] T-K Li and L F Liu ldquoTumor cell death induced by topoi-somerase-targeting drugsrdquoAnnual Review of Pharmacology andToxicology vol 41 pp 53ndash77 2001

[196] Y Pommier E Leo H Zhang and C Marchand ldquoDNA topoi-somerases and their poisoning by anticancer and antibacterialdrugsrdquo Chemistry amp Biology vol 17 no 5 pp 421ndash433 2010

[197] J CWang ldquoCellular roles of DNA topoisomerases a molecularperspectiverdquo Nature Reviews Molecular Cell Biology vol 3 no6 pp 430ndash440 2002

[198] J J Champoux ldquoDNA topoisomerases structure function andmechanismrdquo Annual Review of Biochemistry vol 70 pp 369ndash413 2001

[199] S H Chen N-L Chan and T-S Hsieh ldquoNewmechanistic andfunctional insights into DNA topoisomerasesrdquo Annual Reviewof Biochemistry vol 82 pp 139ndash170 2013

[200] C L Limoli E Giedzinski W M Bonner and J E CleaverldquoUV-induced replication arrest in the xeroderma pigmentosumvariant leads to DNA doublestrand breaks 120574-H2AX formationand Mre11 relocalizationrdquo Proceedings of the National Acadamyof Sciences of the United States of America vol 99 no 1 pp 233ndash238 2002

[201] B Michel S D Ehrlich and M Uzest ldquoDNA double-strandbreaks caused by replication arrestrdquo EMBO Journal vol 16 no2 pp 430ndash438 1997

[202] C Arnaudeau C Lundin andTHelleday ldquoDNAdouble-strandbreaks associated with replication forks are predominantlyrepaired by homologous recombination involving an exchangemechanism in mammalian cellsrdquo Journal of Molecular Biologyvol 307 no 5 pp 1235ndash1245 2001

[203] J-H Lee and T T Paull ldquoATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complexrdquo Sci-ence vol 308 no 5721 pp 551ndash554 2005

[204] K K Khanna and S P Jackson ldquoDNA double-strand breakssignaling repair and the cancer connectionrdquo Nature Geneticsvol 27 no 3 pp 247ndash254 2001

[205] A Montecucco F Zanetta and G Biamonti ldquoMolecular mech-anisms of etoposiderdquo EXCLI Journal vol 14 pp 95ndash108 2015

[206] C Jekimovs E Bolderson A Suraweera M Adams K JOrsquoByrne and D J Richard ldquoChemotherapeutic compoundstargeting the DNA double-strand break repair pathways Thegood the bad and the promisingrdquo Frontiers in Oncology vol 4Article ID Article 86 2014

[207] S J Brill S Dinardo K Voelkel-Meiman and R SternglanzldquoNeed for DNA topoisomerase activity as a swivel for DNAreplication and for transcription of ribosomal RNArdquo Naturevol 326 no 6115 1987

[208] J L Nitiss ldquoDNA topoisomerase II and its growing repertoireof biological functionsrdquoNature Reviews Cancer vol 9 no 5 pp327ndash337 2009

[209] R A Kim and J C Wang ldquoFunction of DNA topoisomerasesas replication swivels in Saccharomyces cerevisiaerdquo Journal ofMolecular Biology vol 208 no 2 pp 257ndash267 1989


[210] O Sundin and A Varshavsky ldquoArrest of segregation leads toaccumulation of highly intertwined catenated dimers Dissec-tion of the final stages of SV40 DNA replicationrdquo Cell vol 25no 3 pp 659ndash669 1981

[211] C Holm T Goto J C Wang and D Botstein ldquoDNA topoiso-merase II is required at the time of mitosis in yeastrdquoCell vol 41no 2 pp 553ndash563 1985

[212] T Uemura H Ohkura Y Adachi K Morino K Shiozakiand M Yanagida ldquoDNA topoisomerase II is required forcondensation and separation of mitotic chromosomes in Spomberdquo Cell vol 50 no 6 pp 917ndash925 1987

[213] S DiNardo K Voelkel andR Sternglanz ldquoDNA topoisomeraseII mutant of Saccharomyces cerevisiae Topoisomerase II isrequired for segregation of daughter molecules at the termina-tion of DNA replicationrdquo Proceedings of the National Acadamyof Sciences of theUnited States of America vol 81 no 9 pp 2616ndash2620 1984

[214] C Holm T Stearns and D Botstein ldquoDNA topoisomerase IImust act at mitosis to prevent nondisjunction and chromosomebreakagerdquoMolecular and Cellular Biology vol 9 no 1 pp 159ndash168 1989

[215] A J Carpenter and A C G Porter ldquoConstruction charac-terization and complementation of a conditional-lethal DNAtopoisomerase II120572 mutant human cell linerdquo Molecular Biologyof the Cell (MBoC) vol 15 no 12 pp 5700ndash5711 2004

[216] L F Liu and J C Wang ldquoSupercoiling of the DNA templateduring transcriptionrdquo Proceedings of the National Acadamy ofSciences of the United States of America vol 84 no 20 pp 7024ndash7027 1987

[217] H-Y Wu S Shyy J C Wang and L F Liu ldquoTranscriptiongenerates positively and negatively supercoiled domains in thetemplaterdquo Cell vol 53 no 3 pp 433ndash440 1988

[218] S J Brill and R Sternglanz ldquoTranscription-dependent DNAsupercoiling in yeastDNA topoisomerasemutantsrdquoCell vol 54no 3 pp 403ndash411 1988

[219] M C Schultz S J Brill Q Ju R Sternglanz and R HReeder ldquoTopoisomerases and yeast rRNA transcription Nega-tive supercoiling stimulates initiation and topoisomerase activ-ity is required for elongationrdquo Genes amp Development vol 6 no7 pp 1332ndash1341 1992

[220] S Ray T Panova G Miller et al ldquoTopoisomerase II120572 promotesactivation of RNApolymerase i transcription by facilitating pre-initiation complex formationrdquo Nature Communications vol 4article 1598 2013

[221] R S Joshi B Pina and J Roca ldquoTopoisomerase II is requiredfor the production of long Pol II gene transcripts in yeastrdquoNucleic Acids Research vol 40 no 16 pp 7907ndash7915 2012

[222] H Bunch B P Lawney Y-F Lin et al ldquoTranscriptionalelongation requires DNA break-induced signallingrdquo NatureCommunications vol 6 Article ID 10191 2015

[223] J A Nickoloff and R J Reynolds ldquoTranscription stimulateshomologous recombination in mammalian cellsrdquo Molecularand Cellular Biology vol 10 no 9 pp 4837ndash4845 1990

[224] S Gonzalez-Barrera M Garcıa-Rubio and A Aguilera ldquoTran-scription and double-strand breaks induce similar mitoticrecombination events in Saccharomyces cerevisiaerdquo Geneticsvol 162 no 2 pp 603ndash614 2002

[225] P Gottipati T N Cassel L Savolainen and T Helle-day ldquoTranscription-associated recombination is dependent onreplication inmammalian cellsrdquoMolecular andCellular Biologyvol 28 no 1 pp 154ndash164 2008

[226] F Aymard B Bugler C K Schmidt et al ldquoTranscriptionallyactive chromatin recruits homologous recombination at DNAdouble-strand breaksrdquo Nature Structural amp Molecular Biologyvol 21 no 4 pp 366ndash374 2014

[227] K W Trotter H A King and T K Archer ldquoGlucocorticoidreceptor transcriptional activation via the BRG1-dependentrecruitment of TOP2120573 and Ku7086rdquo Molecular and CellularBiology vol 35 no 16 pp 2799ndash2817 2015

[228] A E West and M E Greenberg ldquoNeuronal activity-regulatedgene transcription in synapse development and cognitive func-tionrdquo Cold Spring Harbor Perspectives in Biology vol 3 no 6pp 1ndash21 2011

[229] M Sheng andM E Greenberg ldquoThe regulation and function ofc-fos and other immediate early genes in the nervous systemrdquoNeuron vol 4 no 4 pp 477ndash485 1990

[230] J Yang A Bogni E G Schuetz et al ldquoEtoposide pathwayrdquoPharmacogenetics andGenomics vol 19 no 7 pp 552-553 2009

[231] L J Kuo and L-X Yang ldquo120574-H2AX-a novel biomarker for DNAdouble-strand breaksrdquo InVivo (Brooklyn) vol 22 no 3 pp 305ndash309 2008

[232] E Suberbielle P E Sanchez A V Kravitz et al ldquoPhysiologicbrain activity causes DNA double-strand breaks in neuronswith exacerbation by amyloid-120573rdquo Nature Neuroscience vol 16no 5 pp 613ndash621 2013

[233] R Ghirlando and G Felsenfeld ldquoCTCF Making the rightconnectionsrdquo Genes amp Development vol 30 no 8 pp 881ndash8912016

[234] L Uuskula-Reimand H Hou P Samavarchi-Tehrani et alldquoTopoisomerase II beta interacts with cohesin and CTCF attopological domain bordersrdquo Genome Biology vol 17 no 1article 182 2016

[235] A G Newman P Bessa V Tarabykin and P B Singh ldquoActivity-DEPendent Transpositionrdquo EMBO Reports vol 18 no 3 pp346ndash348 2017

[236] A Hendrickx N Pierrot B Tasiaux et al ldquoEpigenetic regula-tions of immediate early genes expression involved in memoryformation by the amyloid precursor protein of alzheimerdiseaserdquo PLoS ONE vol 9 no 6 Article ID e99467 2014

[237] F C Ledesma S F El Khamisy M C Zuma K Osborn andK W Caldecott ldquoA human 51015840-tyrosyl DNA phosphodiesterasethat repairs topoisomerase-mediated DNA damagerdquo Naturevol 461 no 7264 pp 674ndash678 2009

[238] F Gomez-Herreros R Romero-Granados Z Zeng et alldquoTDP2-Dependent Non-Homologous End-Joining Protectsagainst Topoisomerase II-Induced DNA Breaks and GenomeInstability in Cells and In Vivordquo PLoS Genetics vol 9 no 3Article ID e1003226 2013

[239] R Madabhushi L Pan and L-H Tsai ldquoDNA damage and itslinks to neurodegenerationrdquoNeuron vol 83 no 2 pp 266ndash2822014

[240] K S Rao ldquoMechanisms of disease DNA repair defects andneurological diseaserdquo Nature Clinical Practice Neurology vol 3no 3 pp 162ndash172 2007

[241] A M R Taylor and P J Byrd ldquoMolecular pathology of ataxiatelangiectasiardquo Journal of Clinical Pathology vol 58 no 10 pp1009ndash1015 2005

[242] AM R Taylor A Groom and P J Byrd ldquoAtaxia-telangiectasia-like disorder (ATLD) - Its clinical presentation and molecularbasisrdquo DNA Repair vol 3 no 8-9 pp 1219ndash1225 2004

[243] M Fernet M Gribaa M A M Salih M Z Seidahmed J Halland M Koenig ldquoIdentification and functional consequences of


a novel MRE11 mutation affecting 10 Saudi Arabian patientswith the ataxia telangiectasia-like disorderrdquo Human MolecularGenetics vol 14 no 2 pp 307ndash318 2005

[244] G S Stewart R S Maser T Stankovic et al ldquoTheDNA double-strand break repair gene hMRE11 is mutated in individuals withan ataxia-telangiectasia-like disorderrdquo Cell vol 99 no 6 pp577ndash587 1999

[245] The International Nijmegen Breakage Syndrome Study GroupldquoNijmegen breakage syndromerdquo Archives of Disease in Child-hood vol 82 no 5 pp 400ndash406 2000

[246] N Z Borgesius M C deWaard I van der Pluijm et al ldquoAccel-erated age-related cognitive decline and neurodegenerationcaused by deficient DNA repairrdquo The Journal of Neurosciencevol 31 no 35 pp 12543ndash12553 2011

[247] M C De Waard I Van Der Pluijm N Zuiderveen Borgesiuset al ldquoAge-related motor neuron degeneration in DNA repair-deficient Ercc1 micerdquo Acta Neuropathologica vol 120 no 4 pp461ndash475 2010

[248] E Adamec J P Vonsattel and R A Nixon ldquoDNA strand breaksin Alzheimerrsquos diseaserdquo Brain Research vol 849 no 1-2 pp 67ndash77 1999

[249] E Mullaart M E T I Boerrigter R Ravid D F Swaab andJ Vijg ldquoIncreased levels of DNA breaks in cerebral cortex ofalzheimerrsquos disease patientsrdquo Neurobiology of Aging vol 11 no3 pp 169ndash173 1990

[250] M P Murphy and H LeVine ldquoAlzheimerrsquos disease and theamyloid-120573 peptiderdquo Journal of Alzheimerrsquos Disease vol 19 no1 pp 311ndash323 2010

[251] Y Liu and S CWest ldquoDistinct functions of BRCA1 and BRCA2in double-strand break repairrdquo Breast Cancer Research vol 4no 1 pp 9ndash13 2002

[252] J Zhang and S N Powell ldquoThe role of the BRCA1 tumorsuppressor in DNA double-strand break repairrdquo MolecularCancer Research vol 3 no 10 pp 531ndash539 2005

[253] E Suberbielle B Djukic M Evans et al ldquoDNA repair factorBRCA1 depletion occurs in Alzheimer brains and impairscognitive function in micerdquo Nature Communications vol 6article 8897 2015

[254] C-W LamW-L Yeung and C-Y Law ldquoGlobal developmentaldelay and intellectual disability associated with a de novoTOP2B mutationrdquo Clinica Chimica Acta vol 469 pp 63ndash682017

[255] I F King C N Yandava A M Mabb et al ldquoTopoisomerasesfacilitate transcription of long genes linked to autismrdquo Naturevol 501 no 7465 pp 58ndash62 2013

[256] D Marko and F Boege ldquoA possible link between nutritionaluptake of ubiquitous topoisomerase inhibitors and autismrdquoInternational Journal of Developmental Neuroscience vol 53 pp8-9 2016

[257] L Vokalova J Durdiakova and D Ostatnıkova ldquoTopoiso-merases interlink genetic network underlying autismrdquo Interna-tional Journal of Developmental Neuroscience vol 47 pp 361ndash368 2015

[258] S M Gasser T Laroche J Falquet E Boy de la Tour and UK Laemmli ldquoMetaphase chromosome structure Involvementof topoisomerase IIrdquo Journal of Molecular Biology vol 188 no4 pp 613ndash629 1986

[259] W C Earnshaw B Halligan C A CookeMM S Heck and LF Liu ldquoTopoisomerase II is a structural component of mitoticchromosome scaffoldsrdquoThe Journal of Cell Biology vol 100 no5 pp 1706ndash1715 1985

[260] P N Cockerill andW T Garrard ldquoChromosomal loop anchor-age of the kappa immunoglobulin gene occurs next to theenhancer in a region containing topoisomerase II sitesrdquo Cellvol 44 no 2 pp 273ndash282 1986

[261] S V Razin Y S Vassetzky and R Hancock ldquoNuclear matrixattachment regions and topoisomerase II binding and reactionsites in the vicinity of a chicken DNA replication originrdquoBiochemical and Biophysical Research Communications vol 177no 1 pp 265ndash270 1991

[262] M O Christensen M K Larsen H U Barthelmes et alldquoDynamics of humanDNA topoisomerases II120572 and II120573 in livingcellsrdquoThe Journal of Cell Biology vol 157 no 1 pp 31ndash44 2002

[263] C L Peterson ldquoChromatin remodeling enzymes Taming themachines Third in review series on chromatin dynamicsrdquoEMBO Reports vol 3 no 4 pp 319ndash322 2002

[264] Y Adachi M Luke and U K Laemmli ldquoChromosome assem-bly in vitro Topoisomerase II is required for condensationrdquoCell vol 64 no 1 pp 137ndash148 1991

[265] P D Varga-Weisz MWilm E Bonte K DumasMMann andP B Becker ldquoChromatin-remodelling factor CHRAC containsthe ATPases ISWI and topoisomerase IIrdquo Nature vol 388 no6642 pp 598ndash602 1997

[266] E C Dykhuizen D C Hargreaves E L Miller et al ldquoBAFcomplexes facilitate decatenation of DNA by topoisomeraseII120572rdquo Nature vol 497 no 7451 pp 624ndash627 2013

[267] M A Bhat A V Philp D M Glover and H J BellenldquoChromatid segregation at anaphase requires the barren prod-uct a novel chromosome-associated protein that interacts withtopoisomerase IIrdquo Cell vol 87 no 6 pp 1103ndash1114 1996

[268] M P Somma B Fasulo G Siriaco andGCenci ldquoChromosomeCondensation Defects in barren RNA-Interfered DrosophilaCellsrdquo Genetics vol 165 no 3 pp 1607ndash1611 2003

[269] M Durand-Dubief J P Svensson J Persson and K EkwallldquoTopoisomerases chromatin and transcription terminationrdquoTranscription vol 2 no 2 pp 66ndash70 2011

[270] M Durand-Dubief J Persson U Norman E Hartsuiker andK Ekwall ldquoTopoisomerase I regulates open chromatin andcontrols gene expression in vivordquo EMBO Journal vol 29 no13 pp 2126ndash2134 2010

[271] J Walfridsson O Khorosjutina P Matikainen C M Gustafs-son and K Ekwall ldquoA genome-wide role for CHD remodellingfactors and Nap1 in nucleosome disassemblyrdquo EMBO Journalvol 26 no 12 pp 2868ndash2879 2007

[272] F Durrieu K Samejima J M Fortune S Kandels-LewisN Osheroff and W C Earnshaw ldquoDNA topoisomerase II120572interacts with CAD nuclease and is involved in chromatincondensation during apoptotic executionrdquoCurrent Biology vol10 no 15 pp 923ndash926 2000

[273] K Hizume S Araki K Yoshikawa and K Takeyasu ldquoTopoi-somerase II scaffold component promotes chromatin com-paction in a linker-histone H1-dependent mannerrdquo NucleicAcids Research vol 35 no 8 pp 2787ndash2799 2007

[274] F J Iborra H Kimura and P R Cook ldquoThe functionalorganization of mitochondrial genomes in human cellsrdquo BMCBiology vol 2 article 9 2004

[275] S Sobek and F Boege ldquoDNA topoisomerases in mtDNAmaintenance and ageingrdquoExperimental Gerontology vol 56 pp135ndash141 2014

[276] R L Low S Orton and D B Friedman ldquoA truncated form ofDNA topoisomerase II120573 associates with the mtDNA genome inmammalian mitochondriardquo European Journal of Biochemistryvol 270 no 20 pp 4173ndash4186 2003


[277] S Sobek I D Rosa Y Pommier et al ldquoNegative regulation ofmitochondrial transcription bymitochondrial topoisomerase irdquoNucleic Acids Research vol 41 no 21 pp 9848ndash9857 2013

[278] H Zhang Y-W Zhang T Yasukawa I Dalla Rosa S Khiatiand Y Pommier ldquoIncreased negative supercoiling of mtDNAin TOP1mt knockout mice and presence of topoisomerases II120572and II120573 in vertebratemitochondriardquoNucleic Acids Research vol42 no 11 pp 7259ndash7267 2014

[279] H-Z Tsai R-K Lin and T-S Hsieh ldquoDrosophila mitochon-drial topoisomerase III alpha affects the aging process viamaintenance of mitochondrial function and genome integrityrdquoJournal of Biomedical Science vol 23 no 1 article 38 2016

[280] JWu L Feng andT-SHsieh ldquoDrosophila topo III120572 is requiredfor the maintenance of mitochondrial genome and male germ-line stem cellsrdquo Proceedings of the National Acadamy of Sciencesof the United States of America vol 107 no 14 pp 6228ndash62332010

[281] I Trounce E Byrne and SMarzuki ldquoDecline in skeletalmusclemitochondrial respiratory chain function possible factor inageingrdquoThe Lancet vol 333 no 8639 pp 637ndash639 1989

[282] M Corral-Debrinski T Horton M T Lott J M Shoffner MF Beal and D C Wallace ldquoMitochondrial DNA deletions inhuman brain Regional variability and increase with advancedagerdquo Nature Genetics vol 2 no 4 pp 324ndash329 1992

[283] A Bender K J Krishnan C M Morris et al ldquoHigh levels ofmitochondrial DNA deletions in substantia nigra neurons inaging and Parkinson diseaserdquoNature Genetics vol 38 no 5 pp515ndash517 2006

[284] Y Kraytsberg E Kudryavtseva A C McKee C Geula NW Kowall and K Khrapko ldquoMitochondrial DNA deletionsare abundant and cause functional impairment in aged humansubstantia nigra neuronsrdquo Nature Genetics vol 38 no 5 pp518ndash520 2006

[285] K Hattori M Tanaka S Sugiyama et al ldquoAge-dependentincrease in deleted mitochondrial DNA in the human heartPossible contributory factor to presbycardiardquo American HeartJournal vol 121 no 6 pp 1735ndash1742 1991

[286] G A Cortopassi and N Arnheim ldquoDetection of a specificmitochondrial DNA deletion in tissues of older humansrdquoNucleic Acids Research vol 18 no 23 pp 6927ndash6933 1990

[287] T-C Yen K-L King H-C Lee S-H Yeh and Y-HWei ldquoAge-dependent increase of mitochondrial DNA deletions togetherwith lipid peroxides and superoxide dismutase in human livermitochondriardquo Free Radical Biology amp Medicine vol 16 no 2pp 207ndash214 1994

[288] J-H Lin and F J Castora ldquoDNA topoisomerase II frommammalian mitochondria is inhibited by the antitumor drugsm-AMSA and VM-26rdquo Biochemical and Biophysical ResearchCommunications vol 176 no 2 pp 690ndash697 1991

[289] M Fehr G Pahlke J Fritz et al ldquoAlternariol acts as atopoisomerase poison preferentially affecting the II120572 isoformrdquoMolecular Nutrition amp Food Research vol 53 no 4 pp 441ndash4512009

[290] S Soubeyrand L Pope andR J GHache ldquoTopoisomerase II120572-dependent induction of a persistent DNA damage response inresponse to transient etoposide exposurerdquoMolecular Oncologyvol 4 no 1 pp 38ndash51 2010

[291] O J Bandele and N Osheroff ldquoBioflavonoids as poisons ofhuman topoisomerase II120572 and II120573rdquo Biochemistry vol 46 no20 pp 6097ndash6108 2007

[292] M Sabourin and N Osheroff ldquoSensitivity of human typeII topoisomerases to DNA damage Stimulation of enzyme-mediated DNA cleavage by abasic oxidized and alkylatedlesionsrdquo Nucleic Acids Research vol 28 no 9 pp 1947ndash19542000

[293] C Mielke M O Christensen H U Barthelmes and F BoegeldquoEnhanced processing of UVA-irradiated DNA by humantopoisomerase II in living cellsrdquo The Journal of BiologicalChemistry vol 279 no 20 pp 20559ndash20562 2004

[294] S Srivastava and C T Moraes ldquoDouble-strand breaks of mousemuscle mtDNA promote large deletions similar to multiplemtDNA deletions in humansrdquo Human Molecular Genetics vol14 no 7 pp 893ndash902 2005

[295] R E Jensen and P T Englund ldquoNetwork news The replicationof kinetoplast DNArdquoAnnual Review ofMicrobiology vol 66 pp473ndash491 2012

[296] T Melendy C Sheline and D S Ray ldquoLocalization of a typeII DNA topoisomerase to two sites at the periphery of thekinetoplast DNA of Crithidia fasciculatardquoCell vol 55 no 6 pp1083ndash1088 1988

[297] T A Shapiro V A Klein and P T Englund ldquoDrug-promotedcleavage of kinetoplast DNA minicircles Evidence for typeII topoisomerase activity in trypanosome mitochondriardquo TheJournal of Biological Chemistry vol 264 no 7 pp 4173ndash41781989

[298] T A Shapiro and A F Showalter ldquoIn vivo inhibition oftrypanosome mitochondrial topoisomerase II Effects on kine-toplast DNA maxicirclesrdquo Molecular and Cellular Biology vol14 no 9 pp 5891ndash5897 1994

[299] M E Lindsay E Gluenz K Gull and P T Englund ldquoA newfunction of Trypanosoma brucei mitochondrial topoisomeraseII is to maintain kinetoplast DNA network topologyrdquoMolecularMicrobiology vol 70 no 6 pp 1465ndash1476 2008

[300] T A Shapiro ldquoMitochondrial topoisomerase II activity is essen-tial for kinetoplast DNAminicircle segregationrdquoMolecular andCellular Biology vol 14 no 6 pp 3660ndash3667 1994

[301] ZWang andP T Englund ldquoRNA interference of a trypanosometopoisomerase II causes progressive loss of mitochondrialDNArdquo EMBO Journal vol 20 no 17 pp 4674ndash4683 2001

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Signal TransductionJournal of


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Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Despite the dangers inherent in such lesions DSBs areprecisely employed for the intentional but controlled disrup-tion of genomic integrity in biological processes The cellularroles of physiological DSBs can be broadly considered asone of two major functionalities genetic recombination ormanipulation of DNA topology In the former role DSBscan act as genomic ldquoshufflersrdquo carefully but permanentlyrecombining DNA segments for genomic diversificationin lymphocytes [6 7] and germ cells [8] In contrasttopoisomerase-mediated DSBs serve as genomic ldquosculptorsrdquomodulating higher-order DNA structure and serving tofacilitate DNA replication and transcription [9ndash11] regulategene expression [12ndash15] and alter chromatin state [16 17]While this latter form of DSB may at first glance appear tobe little more than a transient intermediate these ldquocleavagecomplexesrdquo are structurally DSBs also exist as longer-livedspecies can be endogenously or exogenously converted intoabortive breaks and are highly spatiotemporally regulatedto both produce functionally diverse genomic contortionsand prevent genotoxicity or failure of genetic transactions[18 19] Hence both sources of DSB will be considered in thisdiscussion

Here we review the wide-ranging biological functions ofthese various physiologicalDSBs and themultilayered regula-tion thereof together constituting cellular ldquoDSB physiologyrdquo(Figure 1) By considering DSB physiology in its variousforms we explore how the cell is able to harness potentiallydestructive DSBs in delicate genetic operations

2 Genetic Recombination DSBs asGenomic Shufflers

21 Physiological DSBs in V(D)J Recombination The roleof physiological DSBs in the diversification of the adaptiveimmune response is well documented V(D)J recombinationdescribes the process whereby lymphoid cells recombine arepertoire of germline variable (V) diversity (D) and joining(J) exon gene segments in various permutations to generateenormous antigen-receptor diversity in immunoglobulins(Ig) and T cell receptors (TCRs) All three exon segments areused for assembling the variable region of the Ig heavy chainand the TCR 120573 and 120575 chains whereas only V and J segmentsare required for the Ig light chain and TCR 120572 and 120574 chains[20]The process involves large genomic rearrangements andDSBs act in these processes as ldquorecombinogenic biomarkersrdquospecifying the recruitment of the recombinatorial machineryIt thus follows that the spatiotemporal regulation of DSBinduction and repair is critical to the precise execution ofthis genetic shuffling and perturbations thereof can producedisease in humans The mechanism proceeds as two broadphases cleavage and joining corresponding to the processesof DSB formation and DSB resolution respectively

211 Cleavage DSB Formation Each V D and J segment isflanked by a recombination signal sequence (RSS) compris-ing conserved heptamer-spacer-nonamer sequence elements[21 22] Cleavage is initiated by the recognition of these RSSelements by the lymphoid-cell specific recombinase complex[23 24] Components of this complex RAG-1 and RAG-2

bind one RSS from each of the paired gene segments to formRAG-RSS complexes associated with each signal sequence[25 26] The two RSS groups are then approximated andsynapsis proceeds to form a precleavage synaptic complex(PcSC)ThePcSC is typically directed between gene segmentswith different RSS spacer lengths (either 12 or 23 base pairs)conforming to the 1223 restriction rule [27] PcSC forma-tion is facilitated by the DNA-bending high-mobility groupproteins HMGB1 and HMGB2 which are widely involved inchromatin architecture [28] Here HMGB12 act to enhanceRAG12 binding to and cleavage of the 23 RSS possibly viastabilisation of RAG-mediated bending of the 23 RSS spacer[29ndash33]The RAG proteins proceed to introduce a nick at thejunction between the RSS heptamer and coding sequenceWhile RAG-1 alone but not RAG-2 possesses DNA-bindingactivity and can mediate signal recognition association ofRAG-1withRAG-2 increases sequence specificity and the sta-bility of the enzyme-DNA complex [34 35] Nick formationis followed by a RAG1-mediated transesterification reactionforming a DSB comprising a blunt 51015840-phosphorylated signalend and a hairpin coding end [23 36] The DSB is thenenclosed in a postcleavage synaptic complex before NHEJrepair in the joining phase [37]

212 Joining DSB Resolution Prior to joining hairpin cod-ing ends are nicked open through the endonuclease activityof the nuclear enzyme Artemis which complexes with theserinethreonine kinase DNA-PKcs which in turn activatesArtemis by phosphorylation [38 39] However studies alsoindicate a postcleavage role for RAG proteins in openinghairpin coding ends through 31015840-flap endonuclease activity[40ndash42] as well as serving as a scaffold to shield theDNA endsfrom aberrant nuclease digestion [43] Modifications duringrecombination are introduced through Artemis-mediatedasymmetric hairpin opening andor terminal deoxynu-cleotidyl transferase- (TdT-) mediated nucleotide additionThe former mechanism enables palindromic repeat or ldquoPrdquonucleotide addition as well as nucleotide loss from codingends [44] while TdT-mediated addition inserts random ldquoNrdquonucleotides at coding ends in a 51015840-to-31015840 direction [38 45]TdT a template-independent polymerase is recruited by acomplex of NHEJ repair proteins comprising XRCC4 DNAligase IV and DNA-PK (itself a complex of Ku70 Ku86and DNA-PKcs) [46ndash49] The subsequent activity of DNApolymerase 120582 and 120583 combined with exonucleases generatescompatible coding ends [50 51] which are then ligated bythe XRCC4-DNA ligase IV-XLF complex to produce therecombined V(D)J product [46 47 52]

213 Regulation of DSBs in V(D)J Recombination An impor-tant point of control in the DSB formation step of V(D)Jrecombination is the cell cycle-dependent regulation ofRAG expression Cyclin A-CDK2 phosphorylation-mediateddegradation of RAG-2 at the G1S transition [53] exerts tem-poral control over RAG-induced DSBs by restricting RAG2expression to the G1 phase of the cell cycle [54] Further-more in p53-deficient mice a nonphosphorylatable T490Amutation of the phosphodegron in the C-terminus of RAG-2 results in increased lymphoma formation characterised by











segregation




V-DJ Joining



selection







Physiological DSBs

DNA

RNA

RNA polymerase

3

5

3

5

3

5

tor

hocytyy e


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Acknowledgments


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Volume 201






















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Volume 2014




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segregation




V-DJ Joining



selection







Physiological DSBs

DNA

RNA

RNA polymerase

3

5

3

5

3

5

tor

hocytyy e


Transcription
















































































Acknowledgments


References


































































































































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












































































Acknowledgments


References


































































































































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































































Acknowledgments


References


































































































































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























































Acknowledgments


References


































































































































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































Acknowledgments


References


































































































































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







































Acknowledgments


References


































































































































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology































Acknowledgments


References


































































































































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















Acknowledgments


References


































































































































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Acknowledgments


References


































































































































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















































































































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















































































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















































































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











































































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

ReviewArticle Physiological Roles of DNA Double-Strand Breaksdownloads.hindawi.com/journals/jna/2017/6439169.pdf · ReviewArticle Physiological Roles of DNA Double-Strand Breaks