Ionizing radiation and genetic risks. XVII. Formation mechanisms underlying naturally occurring DNA deletions in the human genome and their potential relevance for bridging the gap

Mutation Research xxx (2013) xxx–xxx

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MUTREV-8061; No. of Pages 17

Review

Ionizing radiation and genetic risks. XVII. Formation mechanismsunderlying naturally occurring DNA deletions in the human genomeand their potential relevance for bridging the gap between inducedDNA double-strand breaks and deletions in irradiated germ cells

Krishnaswami Sankaranarayanan, Reza Taleei, Shirin Rahmanian, Hooshang Nikjoo *

Radiation Biophysics Group, Department of Oncology-Pathology, Karolinska Instituet, Box 260, Stockholm SE 17176, Sweden

A R T I C L E I N F O

Article history:

Received 5 April 2013

Received in revised form 27 June 2013

Accepted 22 July 2013

Available online xxx

Keywords:

Genetic risk

DNA damage

DNA deletion

DSB repair

NHEJ

NAHR

DSB

A B S T R A C T

While much is known about radiation-induced DNA double-strand breaks (DSBs) and their repair, the

question of how deletions of different sizes arise as a result of the processing of DSBs by the cell’s repair

systems has not been fully answered. In order to bridge this gap between DSBs and deletions, we critically

reviewed published data on mechanisms pertaining to: (a) repair of DNA DSBs (from basic studies in this

area); (b) formation of naturally occurring structural variation (SV) – especially of deletions – in the human

genome (from genomic studies) and (c) radiation-induced mutations and structural chromosomal

aberrations in mammalian somatic cells (from radiation mutagenesis and radiation cytogenetic studies).

The specific aim was to assess the relative importance of the postulated mechanisms in generating

deletions in the human genome and examine whether empirical data on radiation-induced deletions in

mouse germ cells are consistent with predictions of these mechanisms.

The mechanisms include (a) NHEJ, a DSB repair process that does not require any homology and

which functions in all stages of the cell cycle (and is of particular relevance in G0/G1); (b) MMEJ, also a

DSB repair process but which requires microhomology and which presumably functions in all cell cycle

stages; (c) NAHR, a recombination-based DSB repair mechanism which operates in prophase I of meiosis

in germ cells; (d) MMBIR, a microhomology-mediated, replication-based mechanism which operates in

the S phase of the cell cycle, and (e) strand slippage during replication (involved in the origin of small

insertions and deletions (INDELs).

Our analysis permits the inference that, between them, these five mechanisms can explain nearly all

naturally occurring deletions of different sizes identified in the human genome, NAHR and MMBIR being

potentially more versatile in this regard. With respect to radiation-induced deletions, the basic studies

suggest that those arising as a result of the operation of NHEJ/MMEJ processes, as currently formulated,

are expected to be relatively small. However, data on induced mutations in mouse spermatogonial stem

cells (irradiation in G0/G1 phase of the cell cycle and DSB repair presumed to be via NHEJ predominantly)

show that most are associated with deletions of different sizes, some in the megabase range. There is

thus a ‘discrepancy’ between what the basic studies suggest and the empirical observations in

mutagenesis studies. This discrepancy, however, is only an apparent but not a real one. It can be resolved

by considering the issue of deletions in the broader context of and in conjunction with the organization

of chromatin in chromosomes and nuclear architecture, the conceptual framework for which already

exists in studies carried out during the past fifteen years or so. In this paper, we specifically hypothesize

that repair of DSBs induced in chromatin loops may offer a basis to explain the induction of deletions of

different sizes and suggest an approach to test the hypothesis. We emphasize that the bridging of the gap

between induced DSB and resulting deletions of different sizes is critical for current efforts in

computational modeling of genetic risks.

� 2013 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Mutation Research/Reviews in Mutation Research

jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/ rev iew sm rCo mm un i ty ad dr es s : w ww.els evier . co m/lo c ate /mu t r es

* Corresponding author at: Karolinska Instituet, Radiation Biophysics Group, Department of Oncology-Pathology, Box 260, P9-02, Stockholm SE 17176, Sweden.

Tel.: +46 8 5177 2490.

E-mail address: [email protected] (H. Nikjoo).

Please cite this article in press as: K. Sankaranarayanan, et al., Ionizing radiation and genetic risks. XVII. Formation mechanismsunderlying naturally occurring. . .,Mutat. Res.: Rev. Mutat. Res. (2013), http://dx.doi.org/10.1016/j.mrrev.2013.07.003

1383-5742/$ – see front matter � 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.mrrev.2013.07.003


mailto:[email protected]


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K. Sankaranarayanan et al. / Mutation Research xxx (2013) xxx–xxx2

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2. Mechanisms of formation of structural variation in the genome: the roles of DSB repair- and of DNA replication processes . . . . . . . . . . . 000

2.1. Structural variation in the human genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.2. Mechanisms of formation of genomic structural variation: roles of DNA DSB repair and DNA replication processes. . . . . . . . . . . . . 000

2.3. Error-prone DSB repair pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.3.1. ‘Classical’ non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) processes . . . . . . . . . . 000

2.3.2. Non-allelic homologous recombination (NAHR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.3.3. Single-strand annealing (SSA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.4. DNA replication processes in the origin of structural variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.5. Mechanisms in the origin of structural variation in microsatellites, minisatellites, small deletions and insertions (INDELs)

and mobile element insertions (MEIs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.6. Inferences from SV studies of the human genome on the relative contribution of the different formation mechanisms . . . . . . . . . . 000

3. Mechanistic insights on radiation-induced deletions from studies in radiation mutagenesis and radiation cytogenetics . . . . . . . . . . . . . . . 000

3.1. Radiation mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.2. Radiation cytogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

4. Do mechanisms of formation of deletions shed light on the issue of deletion sizes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

5. Chromatin organization in the chromosomes and nuclear architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

5.1. Chromatin organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

5.2. Nuclear architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

6. Computational modeling studies on the interaction of radiation with DNA in chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

7. Outline of the hypothesis on the potential relevance of DSBs occurring or induced in the chromatin loops for the occurrence of deletions

of different sizes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

8. Why is bridging the gap between induced DSBs and deletions important? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

1. Introduction

The concept that most radiation-induced germ cell mutationsare DNA deletions, often encompassing more than one gene, andthat these arise as a result of repair or misrepair of induced DNAdouble strand breaks (DSBs), provides a framework for efforts atestimating genetic risks of exposure to ionizing radiation inhumans in the 21st century. The basic idea that most radiation-induced mutations are deletions, however, is not new: it alreadyemerged from the early genetic analysis of radiation-inducedmutations at specific loci in mouse germ cells in the 1960s and1970s (reviewed in [1]) and arguments to support such a viewhad been advanced even earlier [2]. Cytogenetic and molecularstudies of radiation-induced mutations in mouse germ cells inthe 1980s and 1990s (e.g. [3–10]; reviewed in [11,12]) and inmammalian somatic cells (from the 1980s onwards) (reviewedin [13,14], and more recently in [15]) provided direct evidence forthe deletion nature of the induced mutations.

The deletions analysed in the aforementioned studies werefound to encompass a wide range of sizes—from a few base pairsto a few megabases. Of note here is that the molecular studieswere more often focused on mutants recovered at high radiationdoses (e.g., 6 Gy of X-rays) than at lower doses. At high doses, asignificant proportion of deletions (especially the large ones)could have resulted from two-track events and the question ofwhether large deletions could also be induced at lower doses atwhich single track events predominate remains largely unan-swered. Additionally, the available knowledge at the time thesestudies were performed, did not permit firm inferences onthe mechanism(s) of induction of large and small deletions orenable one to conceptualize the roles of the DNA repair processesassociated with the processing of radiation-induced DSBs (theprincipal radiation-induced molecular lesions of biologicalrelevance) in generating DNA deletions. Answers to thesequestions are of particular relevance for on-going efforts atcomputational modeling of genetic risks of radiation exposures[16–21].

Please cite this article in press as: K. Sankaranarayanan, et al., Ionunderlying naturally occurring. . .,Mutat. Res.: Rev. Mutat. Res. (201

The past two decades have witnessed major advances inknowledge in genome research, cell biology and in theoreticalstudies on interaction of ionizing radiation with DNA in chromatinsome of which are relevant to the present paper. On the genomeresearch side, the advances in question have highlighted the rolesof error-prone DSB repair and of DNA replication-based mecha-nisms in the origin of naturally occurring deletions and otherstructural changes in the genome. As a result, it has becomepossible to extend the basic paradigm in mutation research – thatgene mutations arise as a result of errors in DNA replication and/orrepair – to include chromosomal structural changes as well. On thecell biology side, the advances relate to the organization ofchromatin in the chromosomes and nuclear architecture; theseadvances seem to hold some clues which may enable us to envisionhow small and large deletions might be induced at low radiationdoses. Theoretical (computational modeling) studies on interac-tion of radiation with DNA in chromatin (some accompanied byexperimental studies) carried out during the past 15 years or sohave generated a wealth of data, among others, on the spectra ofDNA damage and frequency distributions of fragments of differentsizes revealing the structural characteristics of the chromatinmodeled in the calculations. We therefore considered it instructivenow to re-visit the questions of the extent of radiation-induceddeletions and the mechanisms which can be postulated to generatethem in germ cell stages of relevance for genetic risk estimation,namely, spermatogonial stem cells (in males) and dictyotene(diplotene) oocytes (in females).

2. Mechanisms of formation of structural variation in thegenome: the roles of DSB repair- and of DNA replicationprocesses

2.1. Structural variation in the human genome

A major theme in current genome research is the study ofstructural variation in the human genome. Structural variation (SV)encompasses unbalanced forms such as deletions, duplications and

izing radiation and genetic risks. XVII. Formation mechanisms3), http://dx.doi.org/10.1016/j.mrrev.2013.07.003


Fig. 1. Non-homologous end joining (NHEJ).

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insertions (which lead to copy number variation, CNV) and balancedforms, namely, inversions and balanced translocations. While theexistence of such structural variation in the genome has been knownfor a long time at the cytogenetic and molecular levels, itsimportance on a genome-wide scale was not fully realized untilthe mid-2000s when the use of genome-scanning technologies ledto the discovery of extensive large-scale SV in the genomes of normal

individuals with no obvious genetic disorders [22,23]. These findingsserved to catalyse interest in studies on the formation mechanismsof genomic structural variation and its role in health and disease.

It is now evident that SVs contribute significantly to normalgenetic variation between individuals and populations. It alsoappears that SVs are more specific to individuals than singlenucleotide polymorphisms [24]. A finite proportion of currentlyknown SVs (especially deletions and duplications) underliegenomic disorders. Further, copy number polymorphisms (CNPs)showing association with specific common diseases are known(reviewed in [25–27]).

The earlier SV/CNV discovery studies used microarray-basedtechnologies and were focused on large deletions and duplicationswhich were mapped to approximate genomic locations [22,23,28].Advances in array technology permitted delineation of smaller SVs[29,30] and advances in sequencing technology have now enabledthe use of sequence-based approaches for mapping SVs at a finescale [31–36]. The data generated through the sequence-basedapproaches are now permitting detailed SV characterization andinferences on the mechanisms of their formation, and theirpotential health impact.

2.2. Mechanisms of formation of genomic structural variation: roles of

DNA DSB repair and DNA replication processes

Our current understanding of the formation mechanisms of SVsin the human genome is largely based upon insights gained overthe years from DNA repair/replication studies conducted usingbacteria, yeast, avian and mammalian somatic cells and fromhuman studies on naturally occurring genomic disorders. Thissynergy has led, among others, to the delineation of (a) twofamilies of DSB repair pathways, namely, non-homologous end-joining and homologous recombination repair (the latter includesnon-allelic homologous recombination and single-strand anneal-ing) [37–44] and (b) a replication-associated process calledmicrohomology-mediated break-induced replication [45–48].Besides, the mechanisms underlying the origin of variable number


of tandem repeat polymorphisms [49–52], small insertions/deletions (INDELs) [53–56] and of mobile element insertions werealready known and have been recently reviewed [57–60].Inferences on the roles of these mechanisms and their relativeimportance in shaping the genome over evolutionary time,however, derive from analyses of the sequence contexts of thebreakpoints of the SVs (‘breakpoint signatures’ which arecharacteristic of each of these processes) and comparisons withan appropriate reference sequence (see [61] for a review).

2.3. Error-prone DSB repair pathways

2.3.1. ‘Classical’ non-homologous end-joining (NHEJ) and

microhomology-mediated end-joining (MMEJ) processes

Non-homologous end-joining (NHEJ), Fig. 1, is one of the twomajor pathways of repair of double-strand breaks (DSBs) (bothspontaneous and radiation-induced) in eukaryotes [42,62]. It canjoin DNA ends with a number of different structures with little orno homology at the junctions. Because of their complexity, mostradiation-induced DSBs cannot be directly ligated, i.e., somelimited processing and gap-filling must take place to get the twoDNA ends into a ligatable configuration. Consequently, smallsequence deletions or additions of nucleotides are introduced atthe junctions during the process (‘‘information scars’’ in theterminology of Lieber [42]) and therefore NHEJ is error-prone atleast in the context of radiation-induced DSBs. Microhomology isnot required although a subset of NHEJ events may use 1–4nucleotide bases at the termini.

The NHEJ pathway, Fig. 1, briefly discussed above has beencalled ‘classical’ or ‘canonical’ pathway. It utilizes the DNA-PK(which includes the Ku70/80 heterodimer and DNA-PKcs) andDNA-ligase IV/XRCC4/XLF complexes and Artemis. However,almost from the time of their initial characterization, cell mutantsfor the canonical NHEJ factors had been recognized in which DSBend-joining occurred with good efficiencies in certain contexts[63–67]. Such end-joining in the absence of canonical NHEJ factorshas been called ‘alternative NHEJ’ (Alt-NHEJ) or ‘back-up’ NHEJ (‘B-NHEJ’)[42,68–71]. Several possibilities can be envisaged and onesuch Alt-NHEJ pathway on which there is considerable currentinterest (although not fully characterized as yet) is what is referredto as microhomology-mediated end-joining (MMEJ, shown inFig. 2). The most important and distinguishing property of MMEJ isthe requirement and use of 5–25 bp microhomology during thealignment of broken ends before joining resulting in deletions



Fig. 2. Microhomology-mediated end joining (MMEJ).


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flanking the original break [70]. This is unlike the situation inclassical NHEJ in which the microhomology at breakpoints isshorter and occur at frequencies expected by chance [72]. It isknown that MMEJ utilizes two proteins that function in single-strand break repair (PARP-1/XRCC1 and Ligase III) [73] and isslower but the details of other proteins involved in MMEJ inmammalian cells have not been fully worked out. Both the classicalNHEJ pathway (Fig. 1), and MMEJ (Fig. 2) function throughout thecell cycle [70]. While many ambiguities remain concerning themechanism and MMEJ was earlier thought of as a back-up repairpathway operating in the absence of the classical NHEJ, it wouldnow seem that MMEJ in mammals is robust, at least in certaincontexts during immune system development [70,71,74]. MMEJ isassociated with non-reciprocal translocations [75].

The presence of ‘‘breakpoint signatures’’ mentioned earlier,short microhomologies at the breakpoint junctions of deletionswith classical NHEJ and somewhat longer microhomologies withMMEJ characterize the modus operandi of NHEJ and MMEJ DSBrepair processes.

2.3.2. Non-allelic homologous recombination (NAHR)

Non-allelic homologous recombination (NAHR) (see Fig. 3)occurs through the same fundamental mechanism in meiosis ashomologous recombination (HR) except that the pairing of thehomologous chromosomes is non-allelic and occurs betweenmisaligned repetitive sequences such as segmental duplications(SDs) that are present in the genome. When this happens,sequences that lie between the repeats that undergo NAHR willbe either deleted or duplicated, thus changing the copy number[43]. Of note here is that NAHR-mediated deletions and duplica-tions will arise when the SDs are in the same orientation. With SDs


in inverted orientation, NAHR can generate inversions [26,76].NAHR between SDs on different chromosomes can lead torecurrent chromosomal translocations [77]. Gu et al. [61] haveprovided an extensive review. Lupski [43] coined the term‘‘genomic disorders’’ for those disorders associated with recurrentlarge-scale deletions and reciprocal duplications that arise via SDmisalignment and NAHR in meiosis (recently reviewed by [25,78].

For NAHR to occur, there must be segments of minimal lengthcalled ‘‘minimal effective processing segments’’ (MEPS) whichshare a high similarity to identity between the participating SDs.The extent of MEPS in meiosis is of the order of 300–500 bp in thecase of duplication/deletion associated with CMT1A/HNPP patients[79]. Most of the structural rearrangements causing genomicdisorders take place between SDs which are 10–400 kb in lengthand have >96% sequence identity [28,43]. In addition to SDs, otherrepetitive sequences which provide the substrate for the occur-rence of NAHR (although less often) include Alu and LINE-1

sequences.Breakpoint analyses of recurrent germline deletions mediated

by NAHR have shown that despite large stretches of sequenceidentity, clustering of breakpoints in the SDs occur suggesting‘hotspots’ for the majority of these recombinations [79–82](reviewed in [83]). DNA structures capable of inducing DSBs suchas palindromic sequences, non-B conformation DNA, minisatellitesand DNA transposons have often been found near the NAHRhotspots [84,85]. NAHR can also occur in somatic cells (see [61] forexamples).

2.3.3. Single-strand annealing (SSA)

SSA (see Fig. 4) is considered as a variant form of HRR. It isinitiated when a DSB occurs (or is induced) between two repeat



Fig. 3. Single strand annealing (SSA).


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sequences oriented in the same direction. Single-stranded regionsare created adjacent to the break, which extend to the repeatedsequences such that the complementary strands can anneal to eachother. This process is facilitated by RPA and RAD52 in a RAD51-independent manner [39,86]. Important to note is that SSA isassociated with loss of sequences between the repeats as well asone of the repeats and so, the process is error-prone. The fact thatthe human genome is replete with repetitive elements such as Alu

prompts the suggestion that SSA may play a role in DSB repair.However, since the Alu elements show sequence diversity andmismatches between the repeat elements can suppress SSA, thisprocess is thought to play only a limited role in DSB repair [39].

2.4. DNA replication processes in the origin of structural variation

While for recurrent deletions/duplications (see Fig. 3a), SD-mediated NAHR provides the best mechanistic explanation, thereare now several examples of non-recurrent and complex SVs whichcould not be readily explained on the basis of NAHR. Thephenomenon was initially reported when studying what wasthought to be non-recurrent duplications at the PLP1 gene (inPelizaeus-Merzbacher Disease, PMD, an X-linked recessive disor-der affecting the central nervous system) [47]. The molecularchanges involve duplication, triplication or deletion of the PLP1

gene with microhomology at the junctions (1–10 bp) that are tooshort to support NAHR. In order to explain the complex nature ofthe rearrangements, Lee et al. proposed the Fork Stalling andTemplate Switching (FoSTeS) model [47]. According to this model(Fig. 5), during DNA replication, the replication fork stalls at oneposition, the lagging strand disengages from the original template


and invades another replication fork and restarts DNA synthesison the new fork priming it via the microhomology between theswitched template site and the original fork. The new templatestrand is not necessarily adjacent to the original replication forkin primary sequence, but probably in 3D physical proximity. Uponannealing, the transferred strand primes its own template-drivenextension at the transferred fork and then anneals. This procedureof disengaging, invading and synthesizing can occur multipletimes. According to the model, two hallmarks characterize FoSTeS-mediated rearrangements: (a) sequence complexity that resultsfrom serial template-driven juxtaposition of sequences fromdifferent genomic locations and (b) sequence homologies at thejunctions [48].

The FoSTeS-model has been further generalized with moremolecular mechanistic details from studies in bacteria, yeast andhumans [87–91] in the microhomology-mediated break-inducedreplication (MMBIR) model. In this model, the rearrangementis initiated by a one-ended DSB resulting from a collapsedreplication fork [45,46,48,92]. Details of the model and examplesof SV-associated disorders consistent with the model arediscussed by Gu et al. [61], Hastings et al. [45], Liu et al. [93],Zhang et al. [92] and Simmons et al. [78] (see also [94]). The datadiscussed by Zhang et al. [92] show that ‘‘. . .the rearrangementsgenerated by FoSTeS/MMBIR can be diverse in scale, from genomicduplications affecting megabases of the human genome to smalldeletions involving a single gene or only one exon’’. In furtherdiscussions, we will use the term MMBIR to include FoSTeS modelas well.

Chen et al. [95–98] examined the breakpoints of smallerDNA rearrangements (between 21 bp up to 10 kb) including



Fig. 4. (a) Non-allelic homologous recombination (NAHR): Duplication and deletion. (b) Non-allelic homologous recombination (NAHR): Inversion. (c) Non-allelic

homologous recombination (NAHR).


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duplications, deletions, insertions and inversions recorded in theHuman Gene Mutation Database (HGMD) [99]. They found thatmany of the rearrangements were complex instead of beingsimple duplications and deletions and proposed what has beenreferred to as the ‘serial replication slippage model’ (SRS) toexplain the observations. It assumes that the 30-end of thenascent strand could dissociate from the original templateand invade other templates on the basis of microhomology.Depending on whether the strand slippage occurs forward orbackward, the nascent strand will have a deletion or duplication.The slippage can occur serially, creating complex rearrange-ment. Of note here is that the SRS model proposed for smallstructural changes shares some general features with theFoSTeS/MMBIR model proposed for the larger changes althoughthere are some differences. These are discussed by Gu et al.[61]. For instance, since the hypothesized slippages take placewithin replication forks, the SRS process may not be able tocause large genomic rearrangements involving hundreds of kbor longer.


2.5. Mechanisms in the origin of structural variation in

microsatellites, minisatellites, small deletions and insertions (INDELs)

and mobile element insertions (MEIs)

Simple sequence repeats (SSRs) – perfect or slightly imperfecttandem repeats of a particular k-mer – constitute a class ofrepetitive sequences comprising about 3% of the human genome.SSRs with a short repeat unit (n = 1–13 bases) are often calledmicrosatellites whereas those with longer repeat units (n = 10–60bases) spanning from about 0.5 kb to several kb are calledminisatellites [100]. The vast majority of microsatellite mutationsrepresent gains and losses of entire repeat units. Slippage duringreplication is a mechanism that has been proposed to explainlength variations in microsatellites [49,101,102]. Mutations atminisatellite loci are attributed to complex gene conversion-likeevents involving recombinational exchanges of repeat unitsbetween alleles [50–52].

Microdeletions and microinsertions of �20 bp constitute animportant type of small-scale structural variation. These are



Fig. 5. Fork stalling and template switching (FoSTeS)/microhomology mediated break induced replication (MMBIR).


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potentially explicable in terms of slippage mutagenesis involvingthe addition or removal of one copy of a mono-, di-, or trinucleotidetandem repeat [53]. Small insertions/deletions (INDELs) in the sizerange of 1–100 bp are abundant in the human genome. The recentdata of Mills et al. [103] show that they constitute about 99% of thealmost 2 million small INDELs (size range: 1–10,000 bp) whichthey identified. From a genome-wide analysis of short (1–100 bp)INDELs in the human genome, Messer and Arndt [55] concludedthat the majority of the insertions are tandem duplications ofdirectly adjacent sequence segments with conserved polarity. Theysuggest that for the short ones (<5 bp) the underlying molecularprocesses generating them are compatible with unequal crossingover (=NAHR) or replication slippage whereas for a considerablefraction of the longer INDELs (>5 bp), the mechanism might bedifferent.

In a more recent study carried out within the framework ofthe 1000 Genomes Project (1000 GP) Montgomery et al. [56]identified a high quality set of 1.6 million INDELs (here defined asgains or losses of up to 50 bp at a single locus) from 179individuals and examined, among others, the possible mecha-nisms of their origin. They found that polymerase slippage duringreplication could explain upwards of three-quarters of all INDELsincluding nearly all ‘hotspot INDELs’1 and the remainder weremostly simple deletions in complex sequence, but insertions did

1 In order to facilitate stratification of INDELs by mutation rate and mechanism,

the sequence contexts were classified as: homopolymer runs (HR) with runs of 6 or

more identical nucleotides, tandem repeats (TR) characterized by both repeat unit

length and tract length, predicted hotspots (PR), sites of near-repetitive genome

sequence, not annotated as HR or TR and non-repetitive sites (NR) that are those not

classified as HR, TR or PR [56].


occur and these were significantly associated with pseudopalin-dromic sequence features compatible with the fork stalling andtemplate switching (FoSTeS) mechanism most commonly asso-ciated with large structural variations as discussed earlier.Another important finding is that around 45% of the INDELsoccurred in 4% of the genome that was classified as INDEL hotspots.

Retrotransposition (mostly of LINE-1 elements) causes rear-rangements in the genome via RNA-intermediates using what isreferred to as target site-primed reverse transcription (TPRT) [104–106]. In this process (referred to as ‘mobile element insertion’, MEIin the tables of this paper), an RNA copy of the originalretrotransposon is first generated and subsequently reverse-transcribed back into the genome by a reverse transcriptase(reviewed in [107]). During this process, two short stretches ofidentical sequence, termed target site duplications (TSDs) arecreated at both ends of the new insertion. In some cases, genomicdeletions are associated with the insertion events [108,109].

2.6. Inferences from SV studies of the human genome on the relative

contribution of the different formation mechanisms

There have been several investigations which consideredpotential mechanisms of formation of SVs (excluding INDELs)using the criteria such as those discussed in the preceding section.These are summarized in Tables 1, 2, 3A and 3B. Inspection ofTable 1 will reveal that the inferred relative contributions of the SVformation mechanisms are different in the different studies. Forinstance, in the study of Korbel et al. [32], NAHR contribution islower than that in the study of Kidd et al. [110]. This is notunexpected because of differences in the methodologies and/or the



Table 1Illustrative studies on mechanisms of formation of naturally occurring germ line structural variants in the human genome.

Study Scope, strategy and main results Inferred relative contribution (in %) of Comments

NHEJ NAHR VNTR MEI

Korbel et al. [32] Paired-end mapping (PEM) of SVs 3 kb

and larger in 2 human genomes; 1297

SVs (including 853 deletions, 322

insertions and 122 inversions mapped;

15%: >100 kb up to megabase level;

30% <5 kb; 65% <10 kb; breakpoint

sequencing of >200 SVs

56 14 3 27 PEM identifies insertions, deletions (INDELs)

and inversions � 3 kb and larger; technology

used may have less power to map within

duplication and hence the lower inferred NAHR

contribution (Kidd et al. [110]); most MEI due

to LINE-1; VNTR also includes Alu and

Interspersed nuclear element-R

Kidd et al. [110] Cloning and sequencing SVs > 8 kb in

length from 8 human genomes; used

fosmid-based end-sequencing pair

(ESP) method to identify 261 SVs

(includes 98 insertions, 129 deletions

and 34 inversions)

33 48 4 15 SD-mediated NAHR more common than LINE-1

or Alu-mediated ones; MEIs likely to be an

underestimate; enrichment of repetitive DNA

for insertions and deletions

Kidd et al. [111] Developed a resource based on

capillary end sequencing of 13.8 million

clones from 17 human genomes; 1054

large SVs (>5 kb; 589 deletions, 384

insertions and 81 inversions)

52 26 3 19 NHEJ group also includes MMEJ as well as

FoSTeS casesa. Nearly all MEI due to LINE-1

insertions; discovery size thresholds > 5 kb

precluded identification of smaller MEIs arising

from Alu or other MEIs

Conrad et al. [29] Mapping over 11,000 CNVs > 443 bp of

which most had been validated

previously; discovered in 41

individuals; of the 5238 genotyped

CNVs, 77% were deletions, 16%,

duplications and 7% multi-allelic; full

analysis of breakpoint sequencing data

in Conrad et al. [127]; see Table 2)

NA 13.5 11.2 NA CNV formation mechanism dependent on CNV

size; NAHR 7-times more likely than VNTR to be

the underlying mechanism in the largest size

decile; VNTR more likely mechanism in the

bottom decile; two motifs forming non-B DNA

structures over-represented in promoter

regions and at CNV breakpoints

NHEJ, non-homologous end-joining; NAHR, non-allelic homologous recombination; VNTR, variable number of tandem repeats, MEI, mobile element insertion; MMEJ,

microhomology-mediated end-joining (same as B-NHEJ); FoSTeS, fork-stalling and template switching. NA: not ascertained.a Kidd et al. [111] divided the 824 structural variants (excluding the 200 due to MEIs and 30 due to VNTRs from the total of 1054) into two classes: Class 1 with no additional

sequence at the breakpoint junction but with microhomology [three sub-groups: 0 or 1matching nucleotides, 92 SVs attributed to NHEJ; 2–20 matching nucleotides, 297 SVs,

NHEJ and MMEJ; 21–100 matching nucleotides, 28 SVs NAHR, other; 101–199 matching nucleotides, 14 SVs, NAHR, other; and �200 matching nucleotides, 233 SV; NAHR.

Class 2 with additional sequence at breakpoints [two sub-groups: 1–10 additional nucleotides, 78, NHEJ; and >10 additional nucleotides, 82, NHEJ, FoSTeS template

switching. In the table above, sub-groups have been pooled appropriately.

Table 3ARelative contributions of the different mechanisms to the formation of human germline deletions in the study of Mills et al. [131] (Based on the authors’ inferences from

‘breakpoint signatures’ summarized in their Fig. 4c and Supplementary Table 11.

Size range Unclassified NAHR NHa,b VNTR MEI Totalc

N % N % N % n % N %

50 bp to > 100 kb 31 0.4 1835 22.4 5088 62.1 843 10.3 401 4.9 8198

a The designation ‘‘non-homologous’’ (NH) is used by the authors to denote absence of extended sequence similarity; under NH are included NHEJ, MMEJ and MMBIR.b Mills (personal communication, March 2013) provided the following additional information: In the initial 10,125 deletion ‘‘call’’s with breakpoints (i.e., including some

SVs for which insertions were the ancestral state), 76.2% had between 2 and 376 bp of homology, 7.7% were blunt-end and 16.1% had non-template sequence insertions.c Represents a subset of SVs (among the total) classified as deletions based on ancestral state.

Table 2Relative contribution of different mechanisms in the formation of human germline copy number variants in the study of Conrad et al. [112]a inferred from ‘breakpoint

signatures’ (from Table 1 of [112]).

Break point signature Possible mechanism(s)b Data required Estimated proportion (%)

>100 bp sequence homology at breakpoints NAHR �100-bp breakpoint resolution 10–15

Tandem repeat array in reference sequence VNTR �100-bp breakpoint resolution 10–15

Blunt ends NHEJ, others? Precise sequence 5

Insertion of <20 bp non-templated sequence but no microhomology NHEJ Precise sequence 20–25

Insertion of >20 bp local sequence MMBIR Precise sequence 5–10

Microhomology (<10 bp) NHEJ, MMEJ, MMBIR Precise sequence 40–50

a Sequencing of breakpoints of all CNVs detected in three individuals (a subset of 41 individuals in the work of Conrad et al. [29]). The authors used targeted hybridization-

based DNA capture and next generation sequencing to characterize breakpoint features. The total number of sequenced breakpoints was 324 of which 315 were deletions. The

sizes of these CNVs encompassed a wider spectrum from 420 bp to 184 kb. The breakpoint locations were estimated at a resolution of 50–100 bp using arrays, then sequencing

300-bp fragments spanning the breakpoints. This Table combines the analysis of array data reported in [29].b NAHR, Non-allelic homologous recombination; VNTR, variable number of tandem repeats; NHEJ, non-homologous end-joining; MMBIR, microhomology-mediated

break-induced replication (represent more complex rearrangements including local microinversions suggesting a replication-based strand switching mechanism); MMEJ,

microhomology-mediated end-joining).


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designs used. Further, large duplications that cannot be spanned bythe PEM (�3 kb) and ESP (up to 40 kb) methods will be under-represented and as Gu et al. [61] note, complex genomicrearrangements may not readily be detected.


In the work of Kidd et al. [111], as footnote 1 shows, an inferred33.4% of the variants are due to NAHR, 20.6% due to NHEJ, 36% dueto NHEJ + MMEJ, and 10.0% due to NHEJ + FoSTeS. Note that thesefigures are slightly different because here the total number used



Table 3BRelative contributions of different mechanisms to the formation of germline deletions of different sizes in the study of Mills et al. [131]. Based on authors’ inferences from

‘breakpoint signatures’ summarized in their Fig. 4c and supplementary Table 11.

Size range Unclassified NAHR NH VNTR MEIa Total

n % n % n % n % n %

50–100 bp 5 - 228 13.8 1136 68.6 262 15.8 26 1.6 1657

101–1000 bp 11 0.3 979 27.3 1845 51.4 446 12.4 306 8.5 3587

1–10 kb 11 0.4 499 20.1 1799 72.4 106 4.3 69 2.8 2484

10–100 kb 1 - 90 25.7 231 66.0 28 8.0 0 0 350

>100 kb 3 2.5 39 32.5 77 64.2 1 0.8 0 0 120

Total 8198

a When relating SV formation to the variant size spectrum, Mills et al. [131] observed marked insertion peaks for MEIs at 300 bp, corresponding to Alu elements, and at 6 kb,

corresponding to L1 class of long interspersed elements (LINEs) (see Fig. 4d of [131]).


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(n = 824) excludes the 200 due to MEIs and 30 due to VNTRswhereas the estimated percentages shown in Table 1 includesthese. However, the total of 824 includes deletions, otherinsertions and inversions and therefore these data do not lendthemselves to easy comparisons with those reported in the studiesof Conrad et al. [29,112] in which the predominant SVs aredeletions. It is of interest to note that a proportion of NAHR eventsutilize LINE-1 and Alu sequences [110] and the large SVs(predominantly deletions) arise via NAHR/SD than via VNTR [29].

Table 2 presents detailed results of Conrad et al. [112] on therelative contributions of the different SV formation mechanisms.As can be noted, some ‘breakpoint signatures’ have been assignedto more than one mechanism. This is because of the fact thatdifferent mutational mechanisms can generate similar breakpointsignatures. For example, MMEJ, MMBIR and NHEJ are all capable ofgenerating deletions with microhomology at the breakpoints andprecise characterizations of the differences in the genome studieshave not yet been possible. Besides the recorded roles of NAHR,NHEJ and VNTR, the overall inference from this Table is that therelative proportions of microhomology- mediated deletions (i.e.,contribution of MMEJ, MMBIR primarily) appear higher comparedto those of other mechanisms. Although this inference is notunambiguously reflected in Table 2, support for this inferencecomes from the discussion of the data in which the authors pointout (a) presence of microhomology of 1-30 base pairs at the ends of219 out of 315 sequenced deletion breakpoints (�70%) which isconsistent with a microhomology-mediated process such asMMBIR or MMEJ and (b) presence of 1–367 bp of insertedsequences at 103 out of 315 breakpoints (�30%), often sequencesfrom the genomic vicinity, in addition to the deletion, consistentwith a replication-based mechanism involving local templateswitching such as MMBIR.

Other points of interest (not shown in Table 2) include first,absence of any correlation between the size of the deletion and therelative contribution of inferred mechanism(s); in part this mightbe due to the fact that although the majority of the 324 CNVbreakpoints pertained to deletions whose sizes ranged from 420 bpto 184 kb, the technique used by the authors was biased towardssmaller events and only 8% of the sequenced breakpoints werefrom larger CNVs > 10 kb. Second, there was no correlationbetween deletion size and the number of inserted bases at thebreakpoint or the length of microhomology. The basis for theseobservations is not clear.

Within the framework of the 1000 Genomes Project (1000 GP)Mills et al. [113] mapped SVs �50 base pairs, based on wholegenome DNA sequencing from 185 human genomes, integratingevidence from complementary SV discovery approaches withextensive experimental validations. The emphasis was on dele-tions (a variant class often associated with disease) with less focuson insertions and duplications. Information on balanced SVs (suchas inversions) was not collected.


Mills et al. [113] initially identified SVs as deletions, tandemduplications, novel sequence insertions and MEIs relative to thehuman reference. Subsequent comparative analyses involvingprimate genomes enabled them to classify SVs as deletions,duplications or insertions relative to inferred ancestral genomicloci reflecting mechanisms of SV formation. They have presentedtheir data on deletions, insertions and duplications relating them tothe inferred formation mechanisms in their Fig. 4c and d and insupplementary Table 11 of their publication. Inferences on mecha-nisms were based on the following criteria associated with break-point signatures: NAHR, associated with long sequence similaritystretches around the breakpoints; NH (for ‘non-homologous’),rearrangements in the absence of extended similarity stretches,associated with NHEJ, MMEJ and MMBIR; VNTR, the shrinking orexpansion of variable number of tandem repeats, frequentlyinvolving simple sequences, by slippage, and MEI, the breakpointsoften contain poly (A) sequence and flanking target site duplications.

Tables 3A and 3B (this paper) are based on the informationprovided for deletions in Fig. 4c and supplementary Table 11 ofMills et al. [113] and pertain to 185 genomes as mentioned earlier.Considering Table 3A first, it is obvious that, over the wide range ofsizes from 50 bp to over 100 kb, NH (which includes NHEJ, MMEJand MMBIR) is the dominant mechanism accounting for over 60%of the origin of deletions, followed by NAHR (22%) and VNTR (10%)in that order. This finding is quite similar to those of Conrad et al.(Table 2) discussed earlier.

Concerning the above-mentioned study, Mills (personal com-munication March 2013) made the following additional observa-tions that are relevant for assessing the relative importance of themechanisms grouped together under ‘NH’: (1) It had been hard todistinguish between NHEJ and MMBIR based on breakpointsignatures and this was a primary confounder; (2) The analysisconducted with their deletions may be biased in that it is enrichedfor more ‘clean deletions’ and may have missed more complexevents formed by some mechanisms (i.e., MMBIR) and; (3) In theinitial 10,215 ‘deletion calls’, 76.2% had between 2 and 376 bp ofhomology, 7.7% were blunt-end and 16.1% had non-templateinsertions. These comments provide a basis for assuming that alarge proportion of the deletions grouped under ‘NH’ arose as aresult of microhomology-mediated mechanisms, presumablyMMBIR and MMEJ. If this were true, this finding is in line withthe tentative inference from the work of Conrad et al. [112]discussed earlier. It also implies that the contribution of NHEJmechanism to the origin of deletions in the genome is probablymuch less than that of MMBIR (and MMEJ).

Table 3B which relates deletion size to formation mechanism,shows that (a) deletions of a wide range of sizes occur as a result ofboth NAHR and NH whereas VNTR expansion/shrinkage led tosmall deletions; (b) at every size interval, the relative contributionof NH is higher than that of NAHR (5 times that of NAHR in the 50–100 bp range, and between 1.8 and 2.6 in the other size ranges); (c)



2 Gribble et al. [129] used NGS to determine the DNA sequence structure of

human chromosome 21 in the first transchromosomic mouse line. This mouse line

which carries a freely segregating copy of human chromosome 21 is a model for

human Down syndrome and was created by a process which included irradiation of

human chromosome 21 (200 Gy) and microcell-mediated chromosome transfer

into a mouse embryonic stem cell line. The 41 gross chromosomal rearrangement

breakpoints that were present in the human chromosome were found to be

frequently associated with 1–4 bp microhomologies and were thus consistent with

NHEJ rather than with NAHR (This paper is cited merely to illustrate the power of

NGS. The radiation dose employed in deriving the Tc1 mouse strain is not the kind of

dose one uses in normal mutagenesis experiments!).


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both NAHR and NH are capable of generating larger deletions(>100 kb) with NH being twice as effective as NAHR in this regard.Viewed in conjunction with the new information discussed in thepreceding paragraph, it seems likely that a majority (although notall) of the large deletions in the column headed ‘NH’ arose as aresult of replication errors!

In summary, the data discussed above support the generalconclusion that most of the naturally occurring structural variationin the human genomes can be explained on the basis of one or theother of the known mechanisms, namely, NHEJ, MMEJ, NAHR,FoSTeS/MMBIR, polymerase slippage during replication and MEI.Of these, the role of MEIs (although listed in the tables, since theydo contribute to the naturally occurring SVs), falls outside thescope and context of this paper and will not be further considered.

3. Mechanistic insights on radiation-induced deletions fromstudies in radiation mutagenesis and radiation cytogenetics

3.1. Radiation mutagenesis

The finding that most radiation-induced mutations in bothmouse germ cells and mammalian somatic cells are deletions wasmentioned in Section 1. At the time most of these studies wereconducted, the focus was more on ascertaining the relativeproportions of point mutations and deletions and with the latter,their extent, with the techniques then available (reviewed in[11,13,14,114]). Sequencing of the breakpoints of deletions to gaininsights into potential mechanisms of repair was undertaken onlyrarely. The limited data that are available on this point areconsistent with NHEJ or possibly MMEJ-mediated repair. Forinstance, in the hamster APRT gene, the gamma-ray-induced large(>4 kb) deletion mutations tended to have short direct or invertedrepeat sequences at their breakpoints and fell into regions rich inA–T base pairs [114]. Similarly, analysis of the X-ray or alpha-particle-induced deletion mutants in the HPRT gene in primaryhuman diploid fibroblasts showed evidence for the presence ofshort direct repeats at the breakpoints suggestive of microhomol-ogy-mediated NHEJ [115,116].

3.2. Radiation cytogenetics

From the early days of radiation cytogenetics, the focus of thefield had been on structural chromosomal aberrations, and amongthem, exchange-type aberrations and not on deletions. However,as discussed below, the conceptual framework and DSB repairprocesses associated with radiation-induced exchange-type aber-rations share several features with deletions justifying a briefdiscussion of exchange-type aberrations here.

Savage [117] has provided an excellent review of the theoriesput forth to explain the origin of exchange-type aberrations andthe experiments they catalysed from about the early 1940s untilthe late 1990s. Briefly, the ‘breakage and reunion theory’ whichdominated (and still continues to dominate) much of the thinkingin the field, assumed that for an exchange aberration to occur,breaks induced in two different chromosomes, close together inspace and time (‘proximity effects’) must undergo illegitimaterejoining. The ‘contact first’ theory held, among others, that theprimary effect of radiation was not a break in the chromosome, butan ‘unstable lesion’; if two such lesions come into close contact,they may initiate an exchange mechanism leading to exchange-type aberrations when complete or open breaks when incomplete.

The recognition that DSBs are the principal initial lesions andthat DSB repair processes underlie the formation of chromosomalaberrations enabled the investigators to successfully incorporatethe molecular dimension to their work and launch radiationcytogenetics on a path of phenomenal growth. Several reviews


cover these and other advances in this field until the present (e.g.,[118–127]).

One major concept advanced in early studies in the context ofexchange-type aberrations is that of ‘proximity effects’ mentionedearlier in this section (reviewed in [125]). It has endured the test oftime and still provides a powerful framework for explaining theorigin of not only exchange-type aberrations but also deletions. Forinstance, for interpreting the induction of exchange-type aberra-tions at low radiation doses, cytogeneticists assumed misjoining oftwo chromosome breaks in close proximity and which presumablyarose from the passage of the same ionization track. Thehypothesis to be advanced in this paper that the folding ofchromatin fibres/or chromatin loops may provide a structuralplatform for the induction of deletions of different sizes (byradiation) rests on a similar concept (see next section).

With the discovery that DSBs and their repair underliestructural chromosomal aberrations, it became evident that theDSB repair processes are common to both exchange-type aberra-tions as well as deletions although their relative relevance seemsdifferent. Of the error-prone DSB repair processes discussed earlierin Section 2, NHEJ, MMEJ and possibly SSA have been inferred toplay important roles in radiation-induced exchange-type aberra-tions. As Cornforth [119] observed ‘‘. . .it is conceivable, even likelythat multiple pathways exist in the formation of radiation-inducedexchange aberrations. Even so, the major body of evidenceimplicates a two-hit mechanism as being dominant, similar inconcept to the venerable breakage and reunion theory. At themolecular level, these ideas find consistency with NHEJ or SSA asunderlying recombinational processes, both of which in thecontext of radiogenic exchange formation, are two hit in nature.Confirmation as to which mechanism is preferred will probablyrequire sequencing across the breakpoints of a representativenumber of translocations in cells surviving radiation exposure.’’

Povirk [128] reviewed data on sequence analysis of breakpointjunctions of translocations derived from clinical tumors, from modelexperimental systems and from papillary thyroid carcinomas (PTC)ascertained in patients who sustained radiation exposure in theChernobyl accident. The main findings are the following: (a)translocations primarily reflect mis-joining of the ends of two ormore DSBs; (b) many junctions show significant loss of DNAsequences at the breakpoints suggesting exonucleolytic degradationof DNA ends prior to mis-joining; (c) the level of microhomologyvaried with the system and the agent and in the thyroid carcinomacases, the microhomology at the junctions was no more than 3 bpand (d) overall considered, the general features of the junctionsequences, particularly the high frequency of small terminaldeletions, the apparent splicing of DNA ends at microhomologiesand gap-filling on aligned DSB ends, are consistent with the knownbiochemical properties of the classical NHEJ (possibly also of MMEJ?)and (e) NAHR between repetitive sequences does not appear to be amajor pathway for translocations associated with DSBs.

The possibility that advanced techniques such as the nextgeneration sequencing (NGS) used in genome research can addmore precision to sequence analysis is borne out by the work ofGribble et al. [129].2




G Model


The main message from radiation mutagenesis studies withsomatic cells and radiation cytogenetic studies (also with somaticcells) from the standpoint of mechanisms is that NHEJ might be theprincipal DSB repair mechanism that underlies the origin ofstructural aberrations, including deletions. Mechanisms whichdepend on extensive homologies such as NAHR do not seem to beinvolved.

4. Do mechanisms of formation of deletions shed light on theissue of deletion sizes?

As discussed in Section 2, for naturally occurring deletions, theabove question can be answered with a qualified ‘yes’. NAHR andMMBIR are two mechanisms which can potentially generatedeletions over a wide range of sizes (in the kb > to Mb range) ifcertain requirements are fulfilled. NAHR is an error-prone DSBrepair process, occurs in germ cells of both sexes in prophase 1 ofmeiosis (spermatocytes in males and oocytes in females) as a resultof specific architectural features of the human genome (e.g.,presence of large segments of repetitive DNA such as SDs) whichallow misalignment of homologous chromosomes and unequalcrossing over. The sizes of the recurrent deletions generated byNAHR can range from a few kb to several Mb depending upon,among others, the distance between the SDs. Similarly, MMBIR alsoseems potentially capable of generating deletions of differentlengths, but the deletions are non-recurrent. But MMBIR is not aDSB repair process: it is DNA-replication based and rearrange-ments (most of which are complex) arise as a result of errors in thereplication process. Polymerase slippage during replication cangenerate small deletions. Complex gene conversion-like events(that are assumed to provide the mechanistic basis for VNTRcontractions and expansions) are not expected to produce largedeletions and the findings reported in Table 3B are consistent withthis expectation.

With respect to NHEJ (Fig. 1) and MMEJ (Fig. 2), basic studies(Section 2) suggest that these DSB repair processes, as currentlyformulated, are capable of generating only relatively smalldeletions. Empirical data from human genomic studies, however,support the view that deletions of different sizes do occur naturallyin the human genome and some of them could be due to NHEJ/MMEJ (see Table 3B; we assume here that a proportion of thedeletions, including some medium and large-sized ones, in columnheaded ‘‘NH’’ is due to NHEJ and MMEJ). This is also true ofradiation-induced mutations in mammalian somatic cells and inmouse germ cells. As discussed in Section 1, most inducedmutations in mouse spermatogonial stem cells (most of which arein the G0/G1 phase of the cell cycle at irradiation) are deletions ofdifferent sizes (including some in the megabase range). Taking intoaccount cell cycle phase specificity, one would assume that thesedeletions arose as a result of the operation of NHEJ repair(predominantly). Note that MMBIR and NAHR cannot be invokedbecause of cell cycle phase specificities.

There is therefore a ‘discrepancy’ between the findings from thebasic studies on NHEJ/MMEJ processes (which suggest that thedeletions generated by these are relatively small) and the empiricaldata from radiation mutagenesis studies (which show thatdeletions of different sizes can be generated via these processes).This ‘discrepancy’, however, is only an apparent and not a real one.It can be resolved by taking into account the fact that the issue ofDNA deletions of different sizes involves more than DNA per se andconsidering it in the context of and in conjunction with theorganization of chromatin in chromosomes and nuclear architec-ture. More specifically, we hypothesize that repair of DSBsoccurring or induced in chromatin loops – ubiquitous structuralelements of chromatin and which have been implicated in virtuallyall levels of chromatin organization – may be important in the


formation of DNA deletions of different sizes when processed byNHEJ/MMEJ. The basic concept underlying our hypothesis thatchromatin organization may play an important role in shaping thepatterns and sizes of deletions in the genome after irradiation,however, is not new. This concept lies at the heart of computationalmodeling studies on the interaction of radiation with DNA inchromatin which have been going on for the past more than 15years. Some relevant findings from this research area are discussedin Section 6.

5. Chromatin organization in the chromosomes and nucleararchitecture

5.1. Chromatin organization

Chromatin, the basic material of eukaryotic chromosomes,consists of DNA complexed with basic proteins, notably histones,and acidic proteins. Its overall structure depends on the stage of thecell cycle. During interphase, the chromatin is structurally diffuseto allow access to RNA and DNA polymerases that transcribe andreplicate the DNA. As the cell prepares to divide, i.e., enters mitosisor meiosis, the chromatin packages more tightly to facilitatealignment of the chromosomes at the metaphase plate and theirsegregation during anaphase.

At its most fundamental level, chromatin consists of a repeatingseries of nucleosomal core ‘‘beads’’ separated by linker DNAstrings. Each nucleosome core is made up of an octamer of basichistone proteins (two each, of histones H2A, H2B, H3 and H4)around which a stretch of 146 bp of double-stranded DNA iswrapped around in 1.75 turns [130,131] Adjacent nucleosomes areconnected by histones of the H1 linker class and a short spacerDNA. This elementary ‘beads-on-a string’ fibre of linked nucleo-somes is approximately 10-nm in diameter. It has long beenassumed that the 10-nm fibre, in turn, coils into a 30-nm diameterhelical structure known as the 30-nm fibre [132,133] compactingthe DNA further. It was proposed that that these 30-nm fibres werefurther folded with a regular periodicity into higher order motifs of200–300 nm in mitotic chromosomes. Several models have beenproposed on how this folding can occur, including the one referredto as the ‘radial loop model’ (which is based on histone depletionexperiments) often discussed in text books to describe chromo-some structure [134,135].

At the metaphase stage of cell division, the chromosomes arevery condensed and each chromatid consists of loops of chromatinfibre, containing approximately 75 kb of DNA per loop. The loopsare attached to a central scaffold of non-histone acidic protein,notably topoisomerase II at the so-called scaffold attachmentregions (SARs) which are stretches of several hundred base pairs ofhighly AT-rich DNA. The resulting loop-scaffold complex iscompacted further by coiling to generate a chromatid [136].

New experimental approaches, however, including a combina-tion of cryo-electron microscopy (Cryo-EM) and small angle X-rayscattering (SAXS) used to study human mitotic chromosomes havenow cast doubt on the existence of the 30 nm chromatin fibre[137,138]. Cryo-EM permits observation of frozen, hydratedbiological structures at high resolution without producing theartifacts seen in conventional EM; SAXS detects repeatingstructures in solutions of biological samples and ultra-small X-ray scattering (USAXS) a newly developed type of SAXS, can resolverepeating structures across larger dimensions such as an entirechromosomal region. The new findings show that the mitoticchromosome consists of irregularly arranged 10-nm nucleosomefibres and that regularly folded 30-nm fibres are not present. Thisconclusion is in line with other recent studies that have questionedwhether mammalian chromosomes contain regular 30-nm fibres[139–141]. If chromosomes are devoid of regular 30-nm fibres and




G Model


10-nm fibres are not continuously folded into successively higher-order structures, the question is: how is the chromosome levelcompaction achieved? Both Fussner et al. [141] and Nishino et al.[137] have proposed that chromosome condensation is achievedby packaging of the 10-nm beads-on-a string chromatin fibres in afractal manner (i.e., the same organization repeats itself atdifferent scales). Evidence for a fractal structure of humaninterphase chromosomes has been published [142,143]. Maeshimaet al. [139], Nishino et al. [137] and Fussner et al. [141] furtherpostulate similar irregular folding in chromatin of the majority ofinterphase nuclei. As Hansen [138] notes the new findings wouldfundamentally alter the way we view chromatin fibre packagingwithin chromosomes.

5.2. Nuclear architecture

It is widely accepted that in eukaryotic cells, each chromosomeoccupies a spatially limited, roughly spherical volume referred toas a chromosomal territory (CT) in the interphase nucleus. TheseCTs are non-randomly arranged within the nuclear space [144–152]. The size of each CT is roughly determined by its DNA content,but is also affected by other factors such as its overall transcriptionstatus [153,154]. Chromosome conformation capture assays [155]show intermingling of neighboring CTs [143] and that genes fromneighboring CTs loop out and ‘‘co-cluster’’ with transcriptionmachinery to form three dimensional interactions called activetranscription hubs [156]. Sanyal et al. [157] have now presentedrecent data from the ENCODE project [158] on long-rangeinteractions such as looping, that alter the relative proximitiesof different chromosomal regions in three dimensions and alsoaffect transcription (see also [159]). There is evidence that thethree-dimensional genome architecture contributes to spatialproximity of chromosomal translocation-prone genes in humanhematological malignancies [151,160–163]. Also of note in thiscontext is the work of Nikiforova et al. [164] who found that tworegions of human chromosome 10 (normally separated by 30 Mb)that are inverted in many radiation-induced thyroid tumors(papillary thyroid carcinoma or PTC 1), were actually found to be inclose proximity in the interphase nucleus in 35% of normal thyroidcells. One of these regions contains the RET oncogene and the otherthe H4 gene. They suggest that such a preformed associationbetween the two regions might provide a structural basis for theinduction of a DSB in each of them by a single radiation trackgenerating the ‘intrachange’ (i.e., inversion).

Savage [165] who commented on this paper noted ‘‘. . .theproximity of chromosomal regions involved in the RET-H4

inversion suggests that association of these regions may beimportant in thyroid cell differentiation. If this association is atarget for radiation-induced exchange,. . . then the precision ofresultant breakpoints implies that mere proximity cannot be thesole prerequisite for exchange. Clearly, very complex molecularmachinery is required to align, clamp, and execute precisechromosomal breakage and rejoining’’ and that ‘‘the Nikiforovafindings represent an extreme example of ‘contact first’ theory’’(mentioned in Section 3.2).

6. Computational modeling studies on the interaction ofradiation with DNA in chromatin

Several computational modeling studies have been carried outduring the past more than a decade aimed at elucidating thenature and distribution of the radiation-induced initial damage inthe cell nucleus based on direct physical interactions with variousDNA sites and chemical interactions between radiation-inducedreactive species (e.g., [166–173] and references cited therein).Radiations of different qualities were considered. Further,


experimental validation of some of the predictions from thesestudies have also been made in suitable biological systems whenpossible (e.g., [174,175]). With the exception of the work ofFriedland et al. who incorporated NHEJ repair in their modeling),the other studies mentioned above relied on initial DSBs as thesource of fragments without taking repair into account. Thebiophysical Monte Carlo code PARTRAC (PARticles TRACks) usedfor simulations in these studies calculates DNA damage in cellsbased on the superposition of simulated track structures in liquidwater to an ‘atom-by-atom’ model of DNA at multiple levels oforganization in nuclei from DNA double-helix over nucleosomes,chromatin fiber and its loops, up to chromatin domains andchromosomes. While a review of these studies is beyond the scopeof this paper, the general conclusions of relevance in the presentcontext deserve mentioning: (a) theoretical calculations thatsimulate the indirect and direct effects of radiation on DNAdemonstrate that fragment size distributions are closely related tothe model of chromatin structure that is used and (b) it is possibleto obtain insights into chromatin structure in the living cell bycombining experimental measurements with radiation damageanalysis using a Monte Carlo approach [175].

7. Outline of the hypothesis on the potential relevance of DSBsoccurring or induced in the chromatin loops for the occurrenceof deletions of different sizes

The inferences from human genome studies on structuralvariation that (a) a finite proportion of naturally occurringdeletions presumably arose as a result of NHEJ/MMEJ repair ofnaturally occurring DSBs; (b) these DSBs could have arisen in anyphase of the cell cycle or germ cell stage of either sex and (c) thesizes of many of these deletions exceed those expected on thebasis of how these repair processes are assumed to operate,suggest the need to consider additional factors. One such factor ischromatin organization and in particular, the folding of chromatinfibres and the existence of chromatin loops discussed in Section 5.Chromatin loops have been implicated in nearly all levels ofchromatin organization and function ranging from kb-sized loopsinvolved in the interaction of upstream elements with promotersto giant loops of hundreds of kb in size that might alter the relativeproximities of different regions of a chromosome or differentchromosomes. We postulate that the folding of chromatin fibresand/or chromatin loops may play important roles in thegeneration of naturally occurring and radiation-induced DNAdeletions of different sizes. The basic concept of invoking a loopedstructure for explaining observations such as the ones just cited isnot new. Such a concept has been used before in studies of theinteraction of radiation with DNA in chromatin outlined in thepreceding section and by Sankaranarayanan [13], Bryant [176],Yoshikawa et al. [177], and Nikjoo et al. [178] in different contexts.

The starting point for the hypothesis is the concept that mostnaturally occurring germline deletions (whose origins are not dueto replication errors) represent the end-result of interactionbetween DSBs which originate in the DNA (e.g., as a result ofaction of reactive oxygen species arising from normal metabolicprocesses) and DSB repair processes such as NHEJ. It assumes that afinite proportion of DSBs may occur in the loops in such a positionthat NHEJ processing of them results in excision of the loop andthus an interstitial deletion (It is assumed that this occurrencemust be a low probability event). The size of the deletion will bedependent on the size of the loop and how the processing occurs. Ina radiation scenario, a single track of ionizing radiation passingthrough the loop, say, where the two arms of the loop lie next toeach other, induces a DSB in each of the two arms which, with NHEJprocessing results in an interstitial deletion. Obviously, thequestion of how often such an event occurs is one of probability




G Model


which is radiation-dose dependent. With more than one track, aswill occur at high doses, the situation will be simpler to envisage.

The hypothesis can be tested in computational modelingstudies. In the model of the interphase nucleus often used in suchstudies of interaction of radiation with DNA and repair, the DNA isassumed to take the form of a 30-nm solenoid fibre which isregularly folded to form loops of factories in chromosome domainsin a defined way [166,167,169,179–181]. The computer ‘experi-ment’ calls for irradiation of the model nucleus (with say, sparselyionizing radiation irradiation such as X-rays or gamma rays atdefined doses), assessment of the proportion of the energydeposition events that impacts the loops and generate DSBs, loopexcision via NHEJ repair and determination of the extent of thedeletion. The results should provide some valuable clues on therole of loops and their extent as outlined above. Similar studies canbe conducted with the recent fractal model of the nucleusproposed by Nishino et al. [137] which assumes a 10-nmchromatin fibre that is irregularly folded. Comparison of the resultsshould inform us on which model is more consistent withobservations. Such studies are in progress (Nikjoo et al., 2013,unpublished).

8. Why is bridging the gap between induced DSBs and deletionsimportant?

As stated at the beginning (Section 1) of this paper, the conceptthat most radiation-induced germ cell mutations are DNAdeletions, often encompassing more than one gene, and that thesearise as a result of repair or misrepair of induced DNA DSBs, lies atthe core of current efforts at estimating genetic risks of radiationexposure. However, the gap between induced DSBs and deletions,especially the large deletions, has not been bridged as yet.Computational approach to the estimation of genetic risks requiresa mechanism to bridge this gap that can be modeled in germ cells(stem cell spermatogonia in males and immature oocytes in thefemales). It is generally assumed that most spermatogonial stemcells are in the G0/G1 phase of the cell cycle. Oocytes are arrested indictyotene (diplotene) stage of meiosis I. We need to have preciseinformation on DSB repair mechanism(s) (and on the necessaryadditional factor(s)) for modeling the induction of deletions ofdifferent sizes including large ones in these cell stages. The onlymechanisms that could be considered are NAHR, NHEJ and MMEJin females and NHEJ and MMEJ in males, but as currentlyformulated, the latter two, without the involvement of higherorder chromatin are not sufficient to explain the observations oflarge deletions. We argue that the bridging of the gap betweeninduced DSB and resulting deletions is critical for current efforts incomputational modeling of genetic risks. Only then, there is thepossibility to assess the phenotypic consequences of radiation-induced deletions. If, for instance, the results of studies discussedabove do not shed any light on the issue being investigated, thenthere will be a need to consider other possibilities/alternatives andalso re-examine the premise and the evidence on which theconcept rests and chart a way forward.

9. Conclusions

Experimental studies have provided us with a fund ofknowledge on how naturally occurring and radiation-inducedDSBs in the DNA are processed by the cellular repair processes andtheir fidelity. However, there is a gap in knowledge on how DNAdeletions of different lengths originate as a result of error-proneness in DSB repair intrinsic to some of the repair processes.Analysis of data on structural variation in the human genome fromthe standpoint of mechanisms (i.e., use of information fromanalysis of breakpoints at nucleotide resolution) permit the


inference that six mechanisms have contributed to the formationof structural variation in the genome over evolutionary time: threeof these (namely, NHEJ, MMEJ and NAHR) (Figs. 1–4) are DSB repairprocesses, the fourth and fifth (MMBIR and replication slippage)are replication-related process, and the sixth, insertion of mobileelements. Of these, NAHR and MMBIR have been inferred togenerate deletions over a wide range of sizes. This inference isconsistent with independent data on genomic disorders associatedwith recurrent deletions of different lengths (arising via NAHRmechanism) and other disorders associated with non-recurrentand complex rearrangements (interpreted as arising via MMBIR).However, with respect to NHEJ and MMEJ, the situation is different.Data on structural variation in the human genome suggest that aproportion of deletions of different sizes (up to >100 kb) isinterpretable as arising via NHEJ/MMEJ. Experimental data onradiation-induced mutations in mammalian somatic cells and inmouse stem cell spermatogonia (in which NHEJ is assumed to bethe repair mechanism acting on radiation-induced DSBs) also showthat that the mutations could be associated with deletions ofdifferent sizes including some in the Mb range. However, theserepair processes as currently envisaged are not predicted togenerate large deletions. We have hypothesized that the occur-rence/induction of deletions of different sizes may be associatedwith the chromatin organization in the chromosomes and nucleararchitecture and could be explained on the basis of repair of DSBoccurring or induced in chromatin loops. A computationalapproach at testing the hypothesis is presented. We argue thatthe bridging of the gap between induced DSB and resultingdeletions is critical for progress in current efforts in computationalmodeling of genetic risks.

Acknowledgments

The authors are deeply grateful to Professor Ryan E. Mills,Department of Computational Medicine and Bioinformatics,University of Michigan Medical School, Ann Arbor, MI, USA forgenerously sharing with us the detailed data, additional informa-tion and explanation relating to formation mechanisms ofstructural variation in the human genome discussed in his co-authored paper (Mills et al., 2011, Nature 470: 59-65), ProfessorPenelope Jeggo, Genome Damage and Stability Centre, Universityof Sussex, Brighton, England, and Dr. Albert Pastink, Department ofToxicogenetics, Leiden University Medical Centre, Leiden, TheNetherlands, for fruitful correspondence on DSB repair processes,and the two anonymous referees for their perceptive comments.Work was partially supported by the Swedish Radiation SafetyAuthority (SSM) and Karolinska Institutet.

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G Model


Glossary

1000 GP: The 1000 Genomes Project, McVean, G.A.; Altshuler (Co-Chair), D.M.;Durbin (Co-Chair), R.M.; Abecasis, G.A.R.; Bentley, D.R.; Chakravarti, A.; Clark,A.G.; Donnelly, P. et al. (2012). ‘‘An integrated map of genetic variation from1,092 human genomes’’. Nature 491 (7422): 56–65.

Alu: A class of interspersed repetitive sequences which are present in > 1 millioncopies in the human genome. Full length Alu elements are approximately 300nucleotides long. Details of their properties have been reviewed [107,182–186].

CMT1A: Charcot—Marie Tooth disease, type 1A and HNPP (hereditary neuropathywith liability to pressure palsies) are autosomal dominant neurological condi-tions. CMT1A is associated with a 1.5 Mb duplication in chromosome 17p12 andHNPP, with a deletion of the same size at the same chromosomal region. Theyrepresent reciprocal products of non-allelic homologous recombination be-tween low copy repeats in 17p12.

CNP: Copy number polymorphism, Copy number variant present in greater than 1%frequency in a population and mostly occurring in multiple copy number states[24].

CNV: Copy number variation constitutes a major sub-group of Structural variation(SV) in the genome. Early on, a CNV was defined as a DNA segment that is >1 kbin size and which is present in a variable copy number in comparison to areference genome [187,188]. More recently, Eichler’s group [24] has defined aCNV as ‘‘an imbalance of genomic sequence (>50 bp) that alters the diploidstatus of a particular locus’’.

Complex DSB: Double strand break is defined as two single strand break onopposite strands of DNA separated by a distance d of about 10–20 bp. Whena DSB is accompanied by additional SSB within the distance d, it is defined as acomplex DSB [189].

Cryo-EM: Cryo electron microscopy, to study structure of a specimen in physio-logical condition.

DNA-PK: DNA-dependent protein kinase.DNA-ligase III: One of the enzymes involved in the repair of DNA-single strand

breaks.ESP: End-Sequence pair method, a clone-based genome-sequencing method which

can detect structural variants > 8 kb in length. See Table 1.FoSTeS: Fork Stalling and Template Switching model [47], A DNA-replication based

model developed to explain some of the structural variation in the genome.Genetic risk: The risk of adverse genetic effects caused by induced mutations and

chromosomal abnormalities that are manifest in the descendants.HR (homologous recombination): HRR (homologous recombination repair, HRR). In

classical genetics, the term homologous recombination (HR) is used to describethe process in sexual organisms through which reciprocal exchange of geneticmaterial occurs in prophase I of meiosis. In this process, the paternal andmaternal chromosomes, each made up of two chromatids, pair (synapse) andexchange genetic material. The pairing is precise i.e., allelic. At the molecularlevel, HR is initiated by programmed occurrence of DNA DSBs in meiosis andrepaired by recombination with homologous sequences on a non-sister chro-matid and therefore HR is best described as ‘homologous recombination repair’(HRR). Since HRR entails no loss or gain of genetic material, it is an error-freeDSB repair process. In mammalian somatic cells, HRR operates as a DSB repairprocess, requires a significant degree of homology and is most precise when theidentical sister chromatid is used as the template for repair. The products of theRAD52 epistasis group of genes are involved. HRR functions mainly in the S andG2 phases of the cell cycle when a sister chromatid is available as a template forrepair. HRR function is critical during DNA replication: one-ended replicationforks can arise indirectly when the replication fork collides into an unrepairedSSB. Replication forks may also stall or collapse when they run into certain baselesions and HRR provides a mechanism for accurate repair of such collapsedreplication forks [39,190,191]. This recombination pathway has been calledbreak-induced replication or replication fork repair [192].

INDELs: Small insertions and deletions of DNA sequences in the size range of 1–100 bp.


k-mer: A unique sequence of length k.LINE-1: Long interspersed repetitive sequences, present in, >500,000 copies in the

human genome. Full length LINE-1 elements about 6 kb long. Only about 3000–5000 LINE-1 elements in the genome represent full length elements.

MEI: Mobile element insertion.MEPS: Minimal effective processing segments.Microhomology: A length of perfect matching sequences of one or more base pairs

shared between the proximal and distal reference sequences at breakpointjunctions in genomic studies on formation of structural aberrations. It is animportant hallmark of several mechanisms such as NHEJ, MMEJ, MMBIR [105].

MMBIR: Microhomology-mediated break-induced replication model, a DNA repli-cation based model that is used to explain some of the structural variability inthe human genome.

MMEJ: Microhomology mediated end joining.NAHR: Non-allelic homologous recombination, also called unequal crossing over

occurring between non-allelic but high identity sequences such as segmentalduplications or Alu sequences that generates recurrent rearrangements.

NGS: Next generation sequencing, high-throughput sequencing technologies thatparallelize the sequencing process, producing thousands or millions ofsequences concurrently.

NH: Non-homologous, associated with NHEJ, MMEJ and MMBIR.One track event, two-track event: Terms used in radiobiology to characterize the

action of radiation on chromosomes or genes; one track event refers to a singletrack of electron or an ion traversing cell nucleus. When dose of radiationincreases to a high level the probability of two tracks intersecting the samesegment of DNA within a few base pair increases. Most environmental andradiation therapy exposures are of one-track event types.

PARP-1: Poly[ADP-ribose] polymerase enzyme coded by the PARP-1 gene; has arole in the repair of single-strand DNA breaks.

PEM: Paired-end mapping, a large-scale genome sequencing method which canidentify structural variants that are � 3 kb or larger. See Table 1.

PLP1: (Proteolipid protein gene 1) in Pelizaeus–Merzbacher disease.PMD: Pelizaeus–Merzbacher disease, an X-linked recessive disorder affecting the

central nervous system [47].SARS: Scaffold attachment regions.SAXS: Small angle X-ray scattering, for the study of chromatin structure.SD: Segmental duplications (also called low copy repeat LCRs) are blocks of

interspersed repetitive sequences that range in size from �10 to � 400 kb inlength. Typically, they share a high level (>95%) of sequence identity. Theirgenome-wide content is about 5–6% [23,193].

Solenoid: In physics, the term solenoid is used for a wire coiled on a central axis; inthe context of chromatin structure, it refers to a model on patterns of coiling andcompaction of DNA i.e., how the 10-nm chromatin fibre coils into a 30-nmdiameter helical structure known as the 30-nm fibre [132]. The formation ofsolenoid from nucleosomes can be compared with winding of a cable (DNA) ona spool (nucleosome) and then folding of wrapped spools.

SRS: Model (serial replication slippage model). It is an extension of the classical‘replication slippage model’ originally proposed by Streisinger et al. [194] toexplain the occurrence of frameshift mutations in the bacteriophage T4, name-ly, that these occur in regions of repeats as a consequence of base mispairing(between two repeats) and strand slippage during DNA replication. Subsequentresearch in E. coli provided ample support for this concept [195–197].

SSR: Simple sequence repeats.SV: Structural variation.TPRT: Target site-primed reverse transcription.VNTR: Variable number of tandem repeats.XLF (also known as Cernunnos): Is a core factor for NHEJ repair of DSBs, playing a

unique role in bridging DNA damage sensing and DSB rejoining steps.XRCC-1: X-ray repair cross-complementing protein-1, a DNA repair protein which

interacts with PARP-1 and DNA-ligase III in single-strand break repair.XRCC-4: X-ray repair cross-complementing protein-4, A DA repair protein involved

in NHEJ repair pathway of DSB repair.



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Ionizing radiation and genetic risks. XVII. Formation mechanisms underlying naturally occurring DNA deletions in the human genome and their potential relevance for bridging the gap