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Contents lists available at ScienceDirect

DNA Repair

j ourna l ho me pa ge: www.elsev ier .com/ locate /dnarepai r

he DNA damage response: The omics era and its impact

asper W.J. Derks, Jan H.J. Hoeijmakers, Joris Pothof ∗

epartment of Genetics, Netherlands Toxicogenomics Center, Erasmus University Medical Center, Dr. Molewaterplein 50,015 GE Rotterdam, The Netherlands

r t i c l e i n f o

rticle history:vailable online xxx

eywords:NA damage responseenomicsranscriptomicsext generation sequencing

a b s t r a c t

The emergence of high density technologies monitoring the genome, transcriptome and proteome in rela-tion to genotoxic stress have tremendously enhanced our knowledge on global responses and dynamicsin the DNA damage response, including its relation with cancer and aging. Moreover, ‘-omics’ technolo-gies identified many novel factors, their post-translational modifications, pathways and global responsesin the cellular response to DNA damage. Based on omics, it is currently estimated that thousands ofgene(product)s participate in the DNA damage response, recognizing complex networks that determinecell fate after damage to the most precious cellular molecule, DNA. The development of next generation

roteomics sequencing technology and associated specialized protocols can quantitatively monitor RNA and DNAat unprecedented single nucleotide resolution. In this review we will discuss the contribution of omicstechnologies and in particular next generation sequencing to our understanding of the DNA damageresponse and the future prospective of next generation sequencing, its single cell application and omicsdataset integration in unraveling intricate DNA damage signaling networks.

© 2014 Elsevier B.V. All rights reserved.

. Introduction to ‘-omics’ technologies

Novel technologies and their applications fuel new insightsnd discoveries in any field of molecular life sciences, medicine,olecular epidemiology and biotechnology. One of those revo-

utions represents technologies that monitor a (nearly) completelass of biomolecules in a process of interest. These data-denseechnologies have been designated omics technologies, in whichhe suffix -omics refers to the respective technologies monitor-ng (I) DNA in the context of complete genomes (genomics),II) genome-wide RNA transcript expression levels representinghe transcriptome (transcriptomics), (III) global protein and/orost-translational modifications (PTMs), designated the proteomeproteomics), or (IV) nearly all cellular metabolites, named the

etabolome (metabolomics).The principle of both proteomics and metabolomics relies on

ass differences measured with great accuracy by mass spectrom-try due to protein/metabolite levels or the presence of PTMs.ophisticated and stringent isolation methods of PTMs and sta-

Please cite this article in press as: K.W.J. Derks, et al., The DNA damahttp://dx.doi.org/10.1016/j.dnarep.2014.03.008

le isotope labeling of amino acids allowing quantitative analysisf protein samples have further propelled proteomics technology.he genome and transcriptome have been extensively investigated

∗ Corresponding author.E-mail address: j.pothof@erasmusmc.nl (J. Pothof).

ttp://dx.doi.org/10.1016/j.dnarep.2014.03.008568-7864/© 2014 Elsevier B.V. All rights reserved.

by micro-array technology over the past decade. Micro-arrays arebased on comparative hybridization of fluorescently labeled DNAor cDNA (in case of RNA expression) under stringent conditionsto capture probes (complementary oligonucleotides) printed ona solid surface. This allows the analysis of (tens of) thousands ofmolecules simultaneously, revolutionizing the scale and depth inwhich DNA and RNA could be investigated.

The recent emergence of next generation sequencing (NGS) hasfurther changed the landscape of genome and transcriptome anal-ysis. NGS, also named massive parallel sequencing, can sequencehundreds of millions DNA molecules simultaneously. A singleNGS run can sequence the human genome ∼37 times in 27 h,thereby tremendously facilitating whole genome (re)sequencingprojects and genome analyses such as single nucleotide polymor-phisms (SNP), mutation, insertion/deletion and DNA methylationdetection. In addition, NGS can map protein–DNA and DNA–DNAinteractions at nucleotide resolution. Transcriptomics of largeand small RNAs can be performed by simultaneously sequencingmillions of cDNA molecules. Since NGS does not rely on captureprobe design and their presence on arrays, novel non-coding RNAs,splice variants, post-transcriptional modifications and nascent RNAsynthesis can be quantitatively analyzed. In this review, we will dis-

ge response: The omics era and its impact, DNA Repair (2014),

cuss the contribution of omics technologies to understanding theDNA damage response (DDR), with the emphasis on genomics andtranscriptomics in particular by NGS technologies, and the futureprospective of omics research in the DDR research field.

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Fig. 1. Schematic overview of DNA damage response (DDR). Components of the DDRhave been classified into three steps: sensors, transducers and effectors. Sensorsand transducers consist of proteins and their post-translational modifications. Effec-tors also include microRNAs and gene expression changes by transcription factors.

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. The DNA damage response

It has been estimated that DNA acquires 10,000 of lesions everyay already from endogenous sources alone such as reactive oxy-en species and metabolic products. In addition, several exogenousources also produce DNA lesions, e.g. ultraviolet (UV) light fromhe sun, ionizing radiation and numerous environmental and man-

ade chemicals. DNA lesions can interfere with vital the DNAetabolic processes replication and transcription as well as with

ssociated chromatin reorganization. In contrast to RNA, proteinsnd metabolites, DNA is the only cellular component that cannote replaced upon damage and therefore solely relies on repair.

t is also the largest molecule in the cell, and when paternal andaternal alleles are considered separate, it is unique in most cells.oreover, since DNA is at the top of the informational hierarchy,

nrepaired DNA lesions or incorrectly repaired DNA damage canave lasting consequences [1]. Indeed, incorrect DNA repair results

n mutations, insertions, deletions or chromosomal aberrations,hich eventually lead to cancer development. Many spontaneously

umors as well as hereditary cancer syndromes have defects in DNAepair and response genes, hence illustrating the importance ofaintaining genome integrity. On the other hand, studies in human

rogeroid syndromes and corresponding transgenic mouse modelsndicate that accumulation of unrepaired DNA damage contributesignificantly to aging and numerous age-related pathologies, againointing toward the significant role of DNA damage in health andisease.

To deal with the adverse effects of DNA damage, cells have anrsenal of DNA repair mechanisms, each recognizing and repair-ng its own spectrum of lesions. In addition to DNA repair systems,ell cycle checkpoints are activated that halt cell proliferation torovide a time window to repair. When damage is beyond repair,ell death or cellular senescence, a permanent cell cycle arrest, isnduced to remove the damaged cell from the tissue or to preventt from replicating, with enhanced risk of mutations and cancer.ll DNA repair systems, cell cycle checkpoints and additional path-ays whose activity changes upon DNA damage are collectively

nown as the DDR. It is of utmost importance that the DDR isightly controlled, since there is a delicate balance between incor-ect repair driving carcinogenesis and hyper-activation, inducingpoptosis or senescence that leads to loss of tissue homeostasis,

contributing factor to aging and age-related pathologies [1–4].oreover, the amount and type of DNA lesions, but also context

e.g. cell type, proliferation vs. post-mitotic), determine the cellu-ar outcome of DNA damage signaling. It is therefore not surprisinghat cells have an ingenious DDR that maximizes survival andecides on cell fate. Studies in the last two decades have presented

schematic overview of DDR signaling layers that coordinate theellular response to DNA damage (Fig. 1). The first step involvesetecting DNA lesions by a class of sensor proteins. These sensorsre required for recruiting various factors to the site of damage suchs DNA repair factors, but also transmit a signal to so-called trans-ucer proteins, of which ATM and ATR checkpoint kinases are theost prominent examples. These transducers in turn diversify and

mplify the damage signal to the third layer, which are so-calledffectors, which control the activity of several cellular processesnd pathways, such as cell cycle arrest and apoptosis. Sensor andransducer signaling primarily relies on protein interactions andlterations in protein activity by PTMs such as phosphorylation,biquitination, etc. Several effectors however, are transcription fac-ors, e.g. p53, or microRNAs, which demonstrates that the RNAomponent within the DDR is also essential. While the basic DDR

Please cite this article in press as: K.W.J. Derks, et al., The DNA damahttp://dx.doi.org/10.1016/j.dnarep.2014.03.008

s drawn in Fig. 1 already consists of >100 genes, transcriptomicsnd proteomics have discovered that hundreds of additional pro-eins are targets of checkpoint kinases and more than a thousandenes are differentially expressed upon DNA damage as a result

Both protein and RNA responses are required for cell fate determination after DNAdamage, i.e. repair and checkpoint recovery, cell death, cellular senescence or dif-ferentiation.

of transcription factor/microRNA regulation. Thus, transcriptomicsand proteomics have tremendously expanded our view of the DDR.

3. Proteomics

Mass spectrometry after protein complex isolation has beeninstrumental to identify novel protein–protein interactions andmodifications and boosted various branches of the molecularlife sciences, including DDR research. In addition, specializedproteomics screens dramatically expanded the components andrepertoire of PTM events in the DDR. PTMs are an integral step insignal transduction and within the DDR, including phosphorylation,acetylation, (poly)ADP-ribosylation, ubiquitination, sumoylationand neddylation [5,6]. Since checkpoint kinases ATM and ATR arecentral nodes in the DDR, one of the first proteomics screeningapproaches aimed at identifying target proteins. ATM and ATRphosphorylate S and T residues in target proteins at a conservedSQ or TQ motif. Antibodies specifically raised against these phos-phorylated motifs were used to isolate ATM/ATR target proteinsphosphorylated after DNA damage, which was followed by massspectrometry analysis [7]. Interestingly, more than 500 ATM/ATRtarget proteins were identified, which were not only known tar-gets involved in DNA repair and checkpoint function, but also manyproteins from processes previously not linked to the DDR such asRNA processing factors. Additional proteomics screens identifiednumerous proteins phosphorylated after DNA damage indepen-dent from ATM/ATR [8–10]. These screens together disclose anextensive network of phosphorylation events, crosstalk betweenATM/ATR and several other signal transduction pathways (e.g.insulin/IGF1 receptor signaling) and identified additional effectorsthat control RNA expression programs.

Other PTMs in the context of DNA damage have also beenanalyzed by proteomics, e.g. ubiquitination [11,12], sumoylation[13–15], parylation [16] and acetylation [17]. These screens identi-fied known DNA repair and checkpoint proteins, but also chromatin

ge response: The omics era and its impact, DNA Repair (2014),

remodeling factors and many proteins previously unknown to par-ticipate in the DDR, indicating the complexity of signaling networksin the DDR at the PTM level. It is highly conceivable that PTMsin the DDR exhibit crosstalk to fine-tune the cellular response

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r outcome of DNA damage signaling. The effector protein p53s among the best-studied examples. p53 is not only phosphory-ated at several amino acids, but is also acetylated, ubiquitinated,umoylated, methylated, neddylated, ADP-ribosylated and glyco-ylated at several residues [18]. Therefore, proteomics screens thatuantify multiple PTMs in parallel could unravel such intricateetworks. A multilevel proteomics approach was designed to quan-ify protein phosphorylation, acetylation and abundance in parallel.his study found that the ubiquitination cascade itself is targetedy several phosphorylation events in the DDR [17]. In summary,roteomics contributed enormously to our understanding of theomplex signaling events in the DDR and the prospect of multi-evel PTM proteomics studies will further unravel these elaborateetworks [19,20].

. Transcriptomics

The cellular outcome of DNA damage signaling is for a large partetermined by transcriptional programs controlled by key effectorroteins, including the transcription factor p53 (Fig. 1). Transcrip-ional reprogramming is essential for the execution and outcomef DDR signaling, e.g. transient cell cycle arrest, senescence orpoptosis. Micro-array technology has significantly enhanced ournderstanding of the transcriptional response associated with DNAamage. Countless micro-array-based transcriptomics studies haveeen published to date in which cells/organisms were exposed toNA damage. It is very difficult to compare results between studiesnd extract common transcriptional changes, because most of thesetudies were performed under completely different conditions, e.g.ell type/tissue, dose and time after treatment. Moreover, technicalariation is induced by choice of micro-array platform, normaliza-ion procedure and statistics. Based on all these micro-array studies,e estimate that the expression of up to a few thousand genes is

ltered after DNA damage, depending on dose, agent, cell type, etc.verall conclusions could be that besides p53 several additional

ranscription factors control gene expression after DNA damagend numerous cellular processes and pathways are controlled byhe DDR at the transcriptional level [21,22].

Global gene expression profiling has been very informativeo interpret the role of DNA damage in the complex pro-esses of aging [23–25]. Human accelerated aging syndromesnd corresponding transgenic mouse models with specific DNAepair defects indicated a causal role of DNA damage in aging,hich was based on age-related pathology and aging pheno-

ypes at the cellular and tissue level [24,26]. Micro-array analysisevealed that a large part of the transcriptome of naturally agedild type mice was significantly overlapping with global gene

xpression profiles from accelerated aging mouse models withefects in transcription-coupled DNA repair. This indicates thatranscription-blocking lesions are involved in establishing theging transcriptional landscape. Moreover, these transcriptomicsnalyses revealed the presence of a DNA damage-triggered sur-ival response, which includes suppression of the somato- (growthormone and IGF1), lacto- and thyrotrophic hormonal axes and

nduction of e.g. the anti- oxidant defense. This response resembleshe longevity-promoting response by dietary restriction as seen inranscriptomics, which is constitutive active in long-lived dwarf

utants. Subsequently, micro-arrays generated from cell culturesxposed to UV, which induces transcription-blocking lesions, mim-cked these age-related gene expression profiles including theurvival response, providing further molecular evidence that DNA

Please cite this article in press as: K.W.J. Derks, et al., The DNA damahttp://dx.doi.org/10.1016/j.dnarep.2014.03.008

amage contributes to aging.Although mRNAs are the most studied RNA molecules to

ate, it is becoming apparent that not-for-protein coding (non-oding) RNAs are abundantly present in cells, even more plentiful

PRESSir xxx (2014) xxx–xxx 3

than mRNAs [27,28]. One of the best-studied classes of non-coding RNAs are microRNAs, which are small (∼22 nucleotides)endogenous non-coding RNAs that repress target gene expres-sion by binding to complementary target sites mainly residingin 3′ UTRs, thereby predominantly inducing mRNA degradation[29]. MicroRNA micro-array technology identified several differen-tially regulated microRNAs in response to DNA-damaging agents[30–36]. Based on microRNA array time series a hypothesis waspostulated that in the DDR microRNAs act in between the fast PTMresponse and the relative slower gene transcriptional responsesvia promoter regulation [31,37]. Since a single microRNA can tar-get hundreds of different mRNAs simultaneously, this observationcould provide a mechanism to rapidly alter a complete gene expres-sion program followed by more stable changes at the promoter.Subsequent evidence by microRNA arrays demonstrated that a sig-nificant part of all microRNA expression after DNA damage wascontrolled by ATM and its target KHSRP [38]. Upon DNA damage,ATM phosphorylates KHSRP, which then binds specific primarymicroRNAs from the nuclear pool of primary microRNAs and accel-erates their biogenesis into mature microRNAs. Thus, microRNAsin the DDR are likely effectors that quickly adapt gene expressionprograms. The transcription-independent mechanism of microRNAregulation provides a manner to transiently and rapidly altergene expression upon DNA damage. Importantly, DNA damageresponsive microRNAs are frequently misexpressed in human can-cer, thereby modulating resistance to genotoxic chemotherapy[35,36,39,40].

Transcriptomics by NGS, also designated RNA sequencing, hasidentified an enormous amount of non-coding RNAs, both smalland long originating from exonic, intronic and intergenic regions[41–47]. The overt majority has unknown functions. StandardmRNA sequencing relies on enrichment of poly-adenylated trans-cripts followed by sequencing. Next to known mature and partiallyprocessed RNA species, sequence information also includes lowabundant mRNAs, poly-adenylated long non-coding RNAs and thecorrect representation of splice variants originating from over95% of the multi-exonic genes [48]. Paired-end sequencing inwhich sequencing is performed from both ends of the cDNAfragments also detects gene fusion events [49] important fortumorigenesis [50–53]. Small RNA sequencing relies on the enrich-ment of all RNA species smaller than ∼30 nucleotides. Sequenceinformation not only detects microRNAs, but also their isoforms(isomiRs), not detectable by array technology. IsomiRs are sequencelength modifications of the mature microRNA due to impreciseprecursor cropping or dicing [54] or post-transcriptional addi-tion of nucleotides to the 3′ end by specialized enzymes [55].Besides microRNAs, small RNA sequencing also detects thousandsadditional small RNAs of which most have unknown functions. Fur-thermore, specific protocols have been developed to sequence longnon-coding RNAs [28], isolate chromatin-bound non-coding RNAs[47], strand-specific sequencing to identify antisense transcripts[56] or nascent RNA [57,58] (Fig. 2).

Currently, only few mRNA or small RNA transcriptomics studiesby NGS in relation to the DDR have been published [59–64] in whichthe data analysis was mainly focused on mRNAs or mature microR-NAs. RNA sequencing identified several long non-coding RNAs thatparticipate in the p53 response by regulating cell cycle arrest andapoptosis [65–68]. In another study nascent RNA isolation followedby NGS was performed to monitor the global effect on RNA syn-thesis by camptothecin treatment, which inhibits topoisomerase Ithereby blocking replication and transcription [69]. Camptothecinprimarily affected transcription elongation and withdrawal led to

ge response: The omics era and its impact, DNA Repair (2014),

transcription resumption starting from the 5′-end of genes, whilestalled RNA polymerases in gene bodies did not recover. Recoveryof RNA synthesis was independent of CSB, an essential compo-nent of transcription-coupled repair (TCR), indicating that TCR

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Fig. 2. Overview of next generation sequencing (NGS) methods. NGS protocols depictedabove the dashed line have been developed to investigate DNA. Detection ofDNA–protein (ChIP), DNA–DNA interactions or chromatin conformational changes(3C-sequencing or its derivatives). Nucleotide resolution-mapping of double strandbreaks (BLESS). Whole genome sequencing (DNA-seq) or only protein-codingregions of the genome (exome sequencing). NGS protocols below the dashed linehave been developed to investigate RNA. RNA–protein interactions by immunopre-cipitation of proteins followed by RNA-sequencing (RIP). Protocols that sequenceRR(

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s not involved in the repair of or RNA synthesis recovery fromranscription-blocking Top1 lesions. One of the key advantages ofGS-based transcriptomics is direct sequence information. It was

hown that DICER and DROSHA, components of the microRNA bio-enesis pathway, are essential for the activation of the DDR at theransducer level. RNA products generated by DICER and DROSHAre required to restore DDR activation. NGS demonstrated that DDRctivation requires DICER- and DROSHA-dependent small RNAsriginating from the site of the double strand DNA break [70].aken together, transcriptomics technologies have been extremelyowerful in deciphering alterations in the transcriptome after DNAamage and provided several new insights in the DDR.

. Genomics

NGS especially impacted DNA research in relation to the DDR.lthough DNA micro-arrays have provided valuable information,GS with the capacity to sequence the genome ∼37 times in 27 hata at nucleotide resolution (compared to hybridization-basedicro-array results) dramatically accelerated and quantitatively

mproved genome research associated with DNA damage (Fig. 2,verview NGS technologies). One of the most frequently used appli-ations of whole genome sequencing or exome sequencing, whichnly sequences known coding areas [71], is the identification ofNP/mutations associated with specific genetic traits or genetic dis-ases, which have been performed for numerous human diseases.mportantly, SNPs or defects in human DDR genes have been linkedy these studies to e.g. accelerated ovarian aging [72], karyome-alic interstitial nephritis [73] and UV sensitivity syndrome, the lastnresolved genetic disorder due to deficiency in nucleotide exci-ion repair [74], linking defects in DDR factors to human age-relatedathology.

Evidently, somatic genomic aberrations due to DNA damage, e.g.utations and chromosomal rearrangements, can be resolved byGS at nucleotide resolution. Although this appears logical, this

Please cite this article in press as: K.W.J. Derks, et al., The DNA damahttp://dx.doi.org/10.1016/j.dnarep.2014.03.008

pproach is met with technical limitations due to the random andnfrequent nature of somatic mutations that cannot be separatedrom sequencing errors. These complications were overcome byerforming a sophisticated single cell sequencing approach that

PRESSir xxx (2014) xxx–xxx

rules out these errors and correctly calls somatic mutations by ENUin the Drosophila genome [75]. One potential complication of sin-gle cell and single DNA sequencing may be the fact that damagesmay be present in the original DNA molecule, which cause de novomutations in the sequencing protocol. In addition to mutations,DNA rearrangements are also often masked. This was improved byStrand-seq [76], a single-cell sequencing technique that sequencesthe original parental DNA template strands in daughter cells fol-lowing cell division. Both single cell-sequencing techniques willbe very useful in determining mutation frequencies of genotoxiccompounds, in cancer samples and during aging.

Next to monitoring genetic aberrations, genomics protocolsare valuable tools to study basic DDR biology. Specialized NGSmethods, chromosome conformation capture sequencing (or itsderivatives), analyze nuclear architecture, nucleosome position-ing or the 3D chromosomal interaction landscape [77]. Thissequencing technique has been used to examine whether chro-mosomal translocations in human cancer originate from selectionof random translocations, targeted DNA damage or frequentinteractions between translocation partners [78]. While loca-tion and frequency of recurrent translocations, including thosedriving B-cell malignancies, is due to targeted DNA break for-mation, nuclear organization was identified as the main driverin non-targeted rearrangements [78]. Another application ofchromosome conformation capture sequencing examined distantenhancer elements of the central DDR transcription factor p53,which drives transcriptional programs triggering cell cycle arrestand in a later stage apoptosis or cellular senescence. Genome-wide p53-binding sites were found located far from any knownp53 target gene. Chromosome conformation capture sequenc-ing discovered that these p53-bound enhancer regions interactintra-chromosomally with multiple neighboring genes to conveylong-distance p53-dependent transcription regulation. Moreover,these regions produced p53-dependent enhancer RNAs that areshort RNAs (200–1000 nucleotide long) required for efficienttranscription of target genes [79]. These results illustrate the com-plexity of the DDR in the context of genomic DNA.

Chromatin immunoprecipitation coupled to NGS, ChIPSeq inshort, maps DNA–protein interactions at nucleotide resolution.Using an inducible double strand DNA break (DSB) system, thechromatin landscape of �H2AX around the DSB was mapped andits spreading properties along the damaged chromosome [80,81].Since chromatin remodeling is essential for a proper DDR, this tech-nology could provide complete chromatin maps from the sites ofDNA damage. ChIPSeq is often used to map transcription factorbinding sites. ChIPSeq provided a genome-wide profile of p53-binding sites, which revealed stimulus-specific functions of p53during differentiation and DNA damage [82]. ChIPSeq was also usedto map single strand DNA by targeting Rad52 in fission yeast, whichbinds to single strand DNA formed at DNA lesions [83]. This methodwas applied to identify DNA damage sites in the genome.

Direct detection of DNA damage and mapping its genomic loca-tion could be applied to identify hotspots for DNA damage andanalyze at which locations DNA repair is most (in)effective. Theseapproaches in ChipSeq are often hampered or limited by the choiceof protein and quality of the antibody. Recently, a method has beendeveloped that directly labels DSBs in situ with a linker followed byisolation and NGS [84]. This approach named BLESS (direct in situbreaks labeling, enrichment on streptavidin and next-generationsequencing) maps DSBs at nucleotide resolution. Replication stress-induced DSBs by aphidicolin in human cells identified more than2000 fragile regions that were overrepresented with genes, satel-

ge response: The omics era and its impact, DNA Repair (2014),

lite repeats and frequently rearranged regions found in humancancer. In toto, genomics approaches by NGS constitute impor-tant tools to monitor DDR processes at unprecedented nucleotideresolution.

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. Conclusion and future prospective

The emergence of NGS technology has dramatically enhancedenomics and transcriptomics studies. Current throughput that canandle dozens to hundreds of samples in a reasonable amountf time, quantitative results (absiolute number of sequences perenomic location/RNA species) instead of relative hybridizationignals and single nucleotide resolution will undoubtedly addressany long-standing questions in the DDR and many other research

reas. Several dedicated experimental approaches have been devel-ped that isolate specific DNA, RNA or chromatin species followedy sequencing. Examples at the DNA level are not only the rela-ively general ChIPSeq [85] and chromosome conformation captureequencing methods [77], but also more specific protocols toonitor DSB sites [84] and single cell analysis of mutations or

hromosomal rearrangements [76] (Fig. 2). At the RNA level, its possible to generate quantitative transcriptomes that includeon-coding RNAs, splice variants, nascent RNA production andNA–chromatin interactions [28,47,49,56–58,71] (Fig. 2). We haveeveloped an RNA sequencing method that does not rely on classelection, but monitors all RNA species, large and small, in a sin-le sequence run thereby quantitatively preserving all RNA classes,llowing cross-class comparisons. Upon cisplatin treatment wedentified numerous differentially expressed RNAs undetectable byther methods, but also a specific global repression of the class oficroRNA and microRNA isoforms (Derks et al., submitted). At the

evel of chromatin, specific properties and changes in chromatinan be monitored using antibodies to specific histone modificationsn combination with NGS providing a genome-wide view of epi-enetic alterations after e.g. genotoxic treatment. We expect thateveral additional genomic and transcriptomic methods based onGS will be developed, which open new perspectives in relation to

he DDR.Although a wealth of omics data has been generated, NGS

ethods have not been exhaustively used in relation to DDResearch. Since DNA and RNA are integral parts of the DDR,e expect several significant discoveries based on NGS data.

or example, NGS genomics studies will provide answers onhe 3-dimensional chromosome conformation during DNA repair.ince many DNA–binding proteins and chromatin remodelers areequired for sensing, transducing and repairing damage, detailedNA–protein interaction maps around the site of damage coulde generated by ChIPSeq. With current NGS-based transcriptomicethodologies differential RNA expression including non-coding

NAs upon genotoxic stress can be mapped. Systematic quan-itative screening in time, including non-coding RNAs as well,omparing the transcriptional response between different DNAesions in similar experimental conditions (dose, cell type, etc) willroduce detailed RNA expression landscapes of the DDR. Becauseoth coding and non-coding RNAs are required for cell fate deter-ination after DNA damage, these maps will assist in unraveling

he complex DDR networks and their involvement in cancer andging. Specialized transcriptomics methods can be used to monitorlobal nascent RNA production and chromatin–RNA interactions inelation to the DDR.

Cellular heterogeneity in organs, cancer, aging, but also in cellultures [86], such as different cell types and cell states (e.g.roliferating vs. post-mitotic), can result in “noise” in datasetsue to various responses to genotoxic stress. This could beddressed by single cell analysis. Several advances in singleell genomics and transcriptomics have been made includingutation and chromosomal rearrangement frequency determi-

Please cite this article in press as: K.W.J. Derks, et al., The DNA damahttp://dx.doi.org/10.1016/j.dnarep.2014.03.008

ation [76,87]. These technologies will be useful to understandhe evolution of cancer and metastasis. Furthermore, the rela-ion of stochastic DNA damage in the aging process can beddressed.

[

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Recent advances in omics technologies offered much morecomplete datasets that monitor the behavior of cellular macro-molecules. Intelligent experimental design will allow integration ofgenomics, transcriptomics and proteomics datasets obtained underidentical conditions and provide a holistic view of the complexDDR networks and final cellular outcome of these signaling events.Shifting the focus from single omics datasets to integration of mul-tiple types of omics datasets requires sophisticated systems biologyapproaches and mathematical modeling. Development of datasetintegration and visualization methods is needed to deal with theselarge and complex datasets. In conclusion, omics technologies, theirintegration and comparison to aging and cancer will tremendouslyenhance our knowledge of the DDR and its relation to health anddisease.

Conflict of interest statement

The authors report no conflict of interest.

Acknowledgments

We acknowledge financial support from the European Com-mission FP7-Health-2008-200880, HEALTH-F2-2010-259893 andNational Institute of Health/National Institute of Ageing (1PO1 AG-17242-02), NIEHS (1UO1 ES011044), and the Royal Academy of Artsand Sciences of The Netherlands (ISK/2483/PAH) (academia profes-sorship to JHJH), European Research Council (233424) AdvancedGrant to JHJH, a KWO grant from the Dutch Cancer Society (2011-5030) and The Netherlands Toxicogenomics Center (050-040-203).

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Kasper W.J. Derks is in the Department of Genetics, Netherlands ToxicogenomicsCenter, Erasmus University Medical Center, Dr. Molewaterplein 50, 3015 GE, Rot-terdam, The Netherlands.

Jan H.J. Hoeijmakers is in the Department of Genetics, Netherlands ToxicogenomicsCenter, Erasmus University Medical Center, Dr. Molewaterplein 50, 3015 GE, Rotterdam,

ge response: The omics era and its impact, DNA Repair (2014),

Joris Pothof is in the Department of Genetics, Netherlands Toxicogenomics Center,Erasmus University Medical Center, Dr. Molewaterplein 50, 3015 GE, Rotterdam, TheNetherlands. E-mail: j.pothof@erasmusmc.nl