Life or Death after a Break: What Determines the Choice?
2019; Elsevier BV; Volume: 76; Issue: 2 Linguagem: Inglês
10.1016/j.molcel.2019.08.023
ISSN1097-4164
AutoresLenno Krenning, Jeroen van den Berg, René H. Medema,
Tópico(s)Traumatic Brain Injury Research
ResumoDNA double-strand breaks (DSBs) pose a constant threat to genomic integrity. Such DSBs need to be repaired to preserve homeostasis at both the cellular and organismal levels. Hence, the DNA damage response (DDR) has evolved to repair these lesions and limit their toxicity. The initiation of DNA repair depends on the activation of the DDR, and we know that the strength of DDR signaling may differentially affect cellular viability. However, we do not fully understand what determines the cytotoxicity of a DSB. Recent work has identified genomic location, (in)correct DNA repair pathway usage, and cell-cycle position as contributors to DSB-induced cytotoxicity. In this review, we discuss how these determinants affect cytotoxicity, highlight recent discoveries, and identify open questions that could help to improve our understanding about cell fate decisions after a DNA DSB. DNA double-strand breaks (DSBs) pose a constant threat to genomic integrity. Such DSBs need to be repaired to preserve homeostasis at both the cellular and organismal levels. Hence, the DNA damage response (DDR) has evolved to repair these lesions and limit their toxicity. The initiation of DNA repair depends on the activation of the DDR, and we know that the strength of DDR signaling may differentially affect cellular viability. However, we do not fully understand what determines the cytotoxicity of a DSB. Recent work has identified genomic location, (in)correct DNA repair pathway usage, and cell-cycle position as contributors to DSB-induced cytotoxicity. In this review, we discuss how these determinants affect cytotoxicity, highlight recent discoveries, and identify open questions that could help to improve our understanding about cell fate decisions after a DNA DSB. To prevent the propagation of mutations, cell division should be limited to cells with undamaged genomes. In the event of a DNA double-strand break (DSB), a signaling cascade is activated, known as the DNA damage response (DDR) (Shaltiel et al., 2015Shaltiel I.A. Krenning L. Bruinsma W. Medema R.H. The same, only different - DNA damage checkpoints and their reversal throughout the cell cycle.J. Cell Sci. 2015; 128: 607-620Crossref PubMed Scopus (82) Google Scholar). The DDR initiates DNA repair and halts cell-cycle progression until repair is complete. Successful DNA repair prevents the propagation of a damaged genome, and the importance of DDR proficiency is highlighted by the fact that many cancer predisposition genes fulfill functions within the process of DNA repair and cell-cycle regulation (Rahman, 2014Rahman N. Realizing the promise of cancer predisposition genes.Nature. 2014; 505: 302-308Crossref PubMed Scopus (210) Google Scholar). DSBs can kill cells, but DSBs can also limit or affect cell viability through different means (Figure 1). Still, very little is known about what determines the cytotoxicity of a DSB. For instance, we do not completely understand whether and how individual DSBs differ in their effect on cellular fitness. The induction of DSBs at different genomic loci illustrates that cytotoxicity of a single DSB may differ per genomic locus (van den Berg et al., 2019van den Berg J. Joosten S.E.P. Kim Y. Manjón A.G. Krenning L. Koob L. Feringa F.M. Klompmaker R. van den Broek B. Jalink K. et al.DNA end-resection in highly accessible chromatin produces a toxic break.bioRxiv. 2019; https://doi.org/10.1101/691857Crossref Google Scholar, Caron et al., 2015Caron P. Choudjaye J. Clouaire T. Bugler B. Daburon V. Aguirrebengoa M. Mangeat T. Iacovoni J.S. Álvarez-Quilón A. Cortés-Ledesma F. Legube G. Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks.Cell Rep. 2015; 13: 1598-1609Abstract Full Text Full Text PDF PubMed Google Scholar, Warmerdam et al., 2016Warmerdam D.O. van den Berg J. Medema R.H. Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.Cell Rep. 2016; 14: 2519-2527Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), possibly determined by location-dependent differences in DNA repair (Feringa et al., 2018Feringa F.M. Raaijmakers J.A. Hadders M.A. Vaarting C. Macurek L. Heitink L. Krenning L. Medema R.H. Persistent repair intermediates induce senescence.Nat. Commun. 2018; 9: 3923Crossref PubMed Scopus (2) Google Scholar, Kalousi and Soutoglou, 2016Kalousi A. Soutoglou E. Nuclear compartmentalization of DNA repair.Curr. Opin. Genet. Dev. 2016; 37: 148-157Crossref PubMed Google Scholar). Several DNA repair pathways have evolved to help resolve DSBs, most notably classical non-homologous end joining (c-NHEJ) and homologous recombination (HR), as well as alternative end-joining pathways. We understand most of the mechanisms required for HR and c-NHEJ, and it is clear that both pathways compete to repair a DSB (Setiaputra and Durocher, 2019Setiaputra D. Durocher D. Shieldin - the protector of DNA ends.EMBO Rep. 2019; 20: e47560Crossref PubMed Scopus (0) Google Scholar). Recent studies have identified the local nuclear environment and epigenetic landscape as determinants of DNA repair pathway usage, and the correct location-dependent pathway choice is essential to limit the toxicity of DSBs (van den Berg et al., 2019van den Berg J. Joosten S.E.P. Kim Y. Manjón A.G. Krenning L. Koob L. Feringa F.M. Klompmaker R. van den Broek B. Jalink K. et al.DNA end-resection in highly accessible chromatin produces a toxic break.bioRxiv. 2019; https://doi.org/10.1101/691857Crossref Google Scholar). In addition to location, the moment during the cell cycle at which a DSB occurs influences repair pathway choice and the toxicity of the DNA-damaging event (Barazas et al., 2019Barazas M. Gasparini A. Huang Y. Küçükosmanoğlu A. Annunziato S. Bouwman P. Sol W. Kersbergen A. Proost N. de Korte-Grimmerink R. et al.Radiosensitivity Is an Acquired Vulnerability of PARPi-Resistant BRCA1-Deficient Tumors.Cancer Res. 2019; 79: 452-460Crossref PubMed Scopus (3) Google Scholar, Chao et al., 2017Chao H.X. Poovey C.E. Privette A.A. Grant G.D. Chao H.Y. Cook J.G. Purvis J.E. Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.Cell Syst. 2017; 5: 445-459.e5Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, Feringa et al., 2016Feringa F.M. Krenning L. Koch A. van den Berg J. van den Broek B. Jalink K. Medema R.H. Hypersensitivity to DNA damage in antephase as a safeguard for genome stability.Nat. Commun. 2016; 7: 12618Crossref PubMed Scopus (6) Google Scholar, Krenning et al., 2014Krenning L. Feringa F.M. Shaltiel I.A. van den Berg J. Medema R.H. Transient activation of p53 in G2 phase is sufficient to induce senescence.Mol. Cell. 2014; 55: 59-72Abstract Full Text Full Text PDF PubMed Google Scholar), which affects the outcome of DSB-inducing treatments (Barazas et al., 2019Barazas M. Gasparini A. Huang Y. Küçükosmanoğlu A. Annunziato S. Bouwman P. Sol W. Kersbergen A. Proost N. de Korte-Grimmerink R. et al.Radiosensitivity Is an Acquired Vulnerability of PARPi-Resistant BRCA1-Deficient Tumors.Cancer Res. 2019; 79: 452-460Crossref PubMed Scopus (3) Google Scholar, Ryl et al., 2017Ryl T. Kuchen E.E. Bell E. Shao C. Flórez A.F. Mönke G. Gogolin S. Friedrich M. Lamprecht F. Westermann F. Höfer T. Cell-Cycle Position of Single MYC-Driven Cancer Cells Dictates Their Susceptibility to a Chemotherapeutic Drug.Cell Syst. 2017; 5: 237-250.e8Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Thus, determinants of DSB-induced cytotoxicity are starting to emerge, yet many questions remain. For instance, could differences in repair kinetics differentially engage the DDR? Also, what causes the difference in the cytotoxicity of a DSB at different locations throughout the genome? It is curious that there seems to be a disconnect between the ability of a cell to overcome the initial cell-cycle arrest and the long-term viability of its offspring (Reyes et al., 2018Reyes J. Chen J.-Y. Stewart-Ornstein J. Karhohs K.W. Mock C.S. Lahav G. Fluctuations in p53 Signaling Allow Escape from Cell-Cycle Arrest.Mol. Cell. 2018; 71: 581-591.e5Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). In this review, we outline the determinants of DSB-induced cytotoxicity, highlight recent discoveries, identify open questions, and summarize ways that could help the field resolve these open questions. The ability to swiftly and accurately repair DSBs is essential to limit their toxicity, as functional inactivation of genes required to repair DNA breaks results in high cellular toxicity due to delayed or deleterious DSB repair. For instance, mutation of the proximal signaling kinase ATM, which gives rise to the ataxia telangiectasia syndrome, causes hypersensitivity to DSBs (Taylor et al., 1975Taylor A.M.R.R. Harnden D.G. Arlett C.F. Harcourt S.A. Lehmann A.R. Stevens S. Bridges B.A. Ataxia telangiectasia: a human mutation with abnormal radiation sensitivity.Nature. 1975; 258: 427-429Crossref PubMed Scopus (734) Google Scholar). In addition, the X-ray repair cross-complementing (XRCC) genes were initially identified by determining DSB toxicity induced by their loss (Thompson et al., 1980Thompson L.H. Rubin J.S. Cleaver J.E. Whitmore G.F. Brookman K. A screening method for isolating DNA repair-deficient mutants of CHO cells.Somatic Cell Genet. 1980; 6: 391-405Crossref PubMed Scopus (0) Google Scholar). Only later were the XRCC genes described to promote DNA repair, uncovering their role in limiting DSB-induced cellular toxicity (Kemp et al., 1984Kemp L.M. Sedgwick S.G. Jeggo P.A. X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoining.Mutat. Res. 1984; 132: 189-196Crossref PubMed Scopus (0) Google Scholar). These examples highlight the dependency on DNA repair activities to limit the toxicity of DSBs. The two canonical pathways for DSB repair are HR and c-NHEJ (Figure 2) (Panier and Durocher, 2013Panier S. Durocher D. Push back to respond better: regulatory inhibition of the DNA double-strand break response.Nat. Rev. Mol. Cell Biol. 2013; 14: 661-672Crossref PubMed Scopus (98) Google Scholar), and the loss of genes involved in either HR or c-NHEJ can result in hypersensitivity toward DSBs. The toxicity of DSBs can either be a result of (excessive) damage signaling or can be caused by failures in repair that affect the genome in such a way that the viability of the cell is compromised. Below, we discuss both of these options by reviewing what we presently know about the limitations of DNA repair pathways and their influence on cellular toxicity in response to DSBs. HR is active in S- and G2 phases of the cell cycle and requires the high cyclin-dependent kinase (CDK) activity present during these stages (Hustedt and Durocher, 2016Hustedt N. Durocher D. The control of DNA repair by the cell cycle.Nat. Cell Biol. 2016; 19: 1-9Crossref PubMed Scopus (122) Google Scholar). HR represents an error-free repair mechanism since it uses the homologous sister chromatid as a template, making it hard to envision how the use of this repair pathway can be deleterious to cell viability. Nonetheless, a variety of HR-repair intermediates poses threats to cell viability. The first HR product that affects cell viability is single-stranded DNA (ssDNA). Long stretches of ssDNA, caused by extensive resection, are toxic to cells (Ochs et al., 2016Ochs F. Somyajit K. Altmeyer M. Rask M.B. Lukas J. Lukas C. 53BP1 fosters fidelity of homology-directed DNA repair.Nat. Struct. Mol. Biol. 2016; 23: 714-721Crossref PubMed Google Scholar, Toledo et al., 2013Toledo L.I. Altmeyer M. Rask M.B. Lukas C. Larsen D.H. Povlsen L.K. Bekker-Jensen S. Mailand N. Bartek J. Lukas J. ATR prohibits replication catastrophe by preventing global exhaustion of RPA.Cell. 2013; 155: 1088-1103Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). In addition, defective exchange of RPA for RAD51 increases the toxicity of DNA breaks (Feringa et al., 2018Feringa F.M. Raaijmakers J.A. Hadders M.A. Vaarting C. Macurek L. Heitink L. Krenning L. Medema R.H. Persistent repair intermediates induce senescence.Nat. Commun. 2018; 9: 3923Crossref PubMed Scopus (2) Google Scholar). Both hyperresection and defective Rad51 loading increase ATR signaling, driving cell-cycle exit (Feringa et al., 2018Feringa F.M. Raaijmakers J.A. Hadders M.A. Vaarting C. Macurek L. Heitink L. Krenning L. Medema R.H. Persistent repair intermediates induce senescence.Nat. Commun. 2018; 9: 3923Crossref PubMed Scopus (2) Google Scholar, Ochs et al., 2016Ochs F. Somyajit K. Altmeyer M. Rask M.B. Lukas J. Lukas C. 53BP1 fosters fidelity of homology-directed DNA repair.Nat. Struct. Mol. Biol. 2016; 23: 714-721Crossref PubMed Google Scholar, Toledo et al., 2008Toledo L.I. Murga M. Gutierrez-Martinez P. Soria R. Fernandez-Capetillo O. ATR signaling can drive cells into senescence in the absence of DNA breaks.Genes Dev. 2008; 22: 297-302Crossref PubMed Scopus (115) Google Scholar, Toledo et al., 2013Toledo L.I. Altmeyer M. Rask M.B. Lukas C. Larsen D.H. Povlsen L.K. Bekker-Jensen S. Mailand N. Bartek J. Lukas J. ATR prohibits replication catastrophe by preventing global exhaustion of RPA.Cell. 2013; 155: 1088-1103Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Besides resection intermediates, the DNA repair product itself may be deleterious. HR is subdivided into multiple sub-pathways such as break-induced replication (BIR), synthesis-dependent strand annealing (SDSA), and double Holliday junction (dHJ) resolution and dissolution (Heyer et al., 2010Heyer W.-D. Ehmsen K.T. Liu J. Regulation of homologous recombination in eukaryotes.Annu. Rev. Genet. 2010; 44: 113-139Crossref PubMed Scopus (483) Google Scholar) (Figure 2). It is thought that the most prevalent pathway in somatic cells, SDSA, does not result in deleterious DNA repair products. Conversely, BIR has been described to cause loss-of-heterozygosity and can induce insertions due to template switching. The last step of HR requires the disentanglement of the sister chromatids, which may involve the removal of dHJs (dHJs) by resolvases, instead of the generally used BTR pathway (reviewed in Sarbajna and West, 2014Sarbajna S. West S.C. Holliday junction processing enzymes as guardians of genome stability.Trends Biochem. Sci. 2014; 39: 409-419Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]). However, improper activity of these dHJ resolvases has been suggested to lead to unresolved recombination intermediates and toxicity by causing mitotic errors. Recent data suggest that the activity of these enzymes is critically required to limit formation of ultra-fine bridges and lagging chromosomes (Chan et al., 2018Chan Y.W. Fugger K. West S.C. Unresolved recombination intermediates lead to ultra-fine anaphase bridges, chromosome breaks and aberrations.Nat. Cell Biol. 2018; 20: 92-103Crossref PubMed Scopus (13) Google Scholar). Finally, breaks in the 45S rDNA are toxic when repaired by HR (Warmerdam et al., 2016Warmerdam D.O. van den Berg J. Medema R.H. Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.Cell Rep. 2016; 14: 2519-2527Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) (Figure 3). This HR-associated toxicity is likely caused by inter-repeat recombination, reducing the rDNA copy number and compromising cell viability. In addition, loss of the dead-box helicase DDX21 results in facial deformities, which were shown to be caused by rDNA damage and lack of rDNA transcription (Calo et al., 2015Calo E. Flynn R.A. Martin L. Spitale R.C. Chang H.Y. Wysocka J. RNA helicase DDX21 coordinates transcription and ribosomal RNA processing.Nature. 2015; 518: 249-253Crossref PubMed Scopus (85) Google Scholar). It was suggested that these phenotypes dramatically decrease cellular viability during development. TP53 loss attenuated the developmental defects, implicating DNA damage responses in the development of facial deformities. In conclusion, even though HR promotes error-free DNA repair, the repair process itself creates several liabilities for cell viability. In contrast to HR, c-NHEJ is active throughout interphase (Hustedt and Durocher, 2016Hustedt N. Durocher D. The control of DNA repair by the cell cycle.Nat. Cell Biol. 2016; 19: 1-9Crossref PubMed Scopus (122) Google Scholar). It depends on the ligation of broken DNA ends, resulting in small insertions and deletions (indels), making the process inherently error prone (Burma et al., 2006Burma S. Chen B.P.C. Chen D.J. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity.DNA Repair (Amst.). 2006; 5: 1042-1048Crossref PubMed Scopus (257) Google Scholar) (Figure 2). Detection of a DSB is performed by the Ku70-80 heterodimer, which recognizes and binds to (near) blunt DNA ends. Then, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is recruited, which phosphorylates multiple substrates to prepare for end ligation. Finally, the ligase IV complex (XRCC4, XLF, and DNA ligase IV) is recruited and ligates the broken DNA ends (Burma et al., 2006Burma S. Chen B.P.C. Chen D.J. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity.DNA Repair (Amst.). 2006; 5: 1042-1048Crossref PubMed Scopus (257) Google Scholar). DNA repair by c-NHEJ is rarely problematic for cell viability; however, in some contexts, its activity can be toxicity. For instance, insertion and deletions caused by imprecise c-NHEJ-mediated repair have the potential to cause toxicity when occurring in essential genes (Wang et al., 2015Wang T. Birsoy K. Hughes N.W. Krupczak K.M. Post Y. Wei J.J. Lander E.S. Sabatini D.M. Identification and characterization of essential genes in the human genome.Science. 2015; 350: 1096-1101Crossref PubMed Scopus (752) Google Scholar). The most frequent repair outcomes of c-NHEJ are small insertions and deletions. However, translocations occur throughout the genome as a result of c-NHEJ with a frequency of 2 × 10−4–2 × 10−3 per DSB (Ghezraoui et al., 2014Ghezraoui H. Piganeau M. Renouf B. Renaud J.-B. Sallmyr A. Ruis B. Oh S. Tomkinson A.E. Hendrickson E.A. Giovannangeli C. et al.Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining.Mol. Cell. 2014; 55: 829-842Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Although translocations are rare, they do pose a critical threat to cells harboring them. The fusions of two centromere-containing fragments in particular are exceptionally harmful to cells. Upon entry into mitosis, a single fused chromatid that contains two centromeres can attach to opposite spindle poles, which will trigger a breakage-fusion-bridge cycle, among other forms of genomic instability, that is detrimental to cell viability (extensively reviewed by Soto et al., 2019Soto M. Raaijmakers J.A. Medema R.H. Consequences of Genomic Diversification Induced by Segregation Errors.Trends Genet. 2019; 35: 279-291Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar]). During normal physiology, telomeres are capped by the specialized protein complex Shelterin (Palm and de Lange, 2008Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1102) Google Scholar). However, every round of DNA replication shortens telomeres to the point at which Shelterin can no longer cap the chromosome ends. Subsequently, critically shortened telomeres are recognized as DNA breaks due to structural similarity and are subjected to the activities of DNA repair pathways (Palm and de Lange, 2008Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1102) Google Scholar). The activation of c-NHEJ, in particular, results in problematic end-to-end fusions of chromosomes (Smogorzewska et al., 2002Smogorzewska A. Karlseder J. Holtgreve-Grez H. Jauch A. de Lange T. DNA ligase IV-dependent NHEJ of deprotected mammalian telomeres in G1 and G2.Curr. Biol. 2002; 12: 1635-1644Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), which can no longer be properly segregated during mitosis (Figure 3). Despite the negative effects of c-NHEJ on cell viability, it remains the most efficient and most frequently used DSB repair pathway in mammalian cells (Löbrich and Jeggo, 2017Löbrich M. Jeggo P. A Process of Resection-Dependent Nonhomologous End Joining Involving the Goddess Artemis.Trends Biochem. Sci. 2017; 42: 690-701Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). c-NHEJ clears the vast majority of DSBs; therefore, this DNA repair pathway is the most critical for limiting the toxicity of DNA breaks. Nonetheless, when c-NHEJ is used in essential sequences such as genes, regulatory regions, and telomeres, c-NHEJ can be a detrimental process. A variety of backup pathways are in place to deal with DSBs that cannot be repaired by means of HR or c-NHEJ (Figure 2) (Ceccaldi et al., 2016Ceccaldi R. Rondinelli B. D’Andrea A.D. Repair Pathway Choices and Consequences at the Double-Strand Break.Trends Cell Biol. 2016; 26: 52-64Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). These pathways rely on end resection and can be dependent on the presence of microhomologies that are used to anneal the broken ends. For example, microhomology-mediated end joining (MMEJ), or alternative end-joining (alt-EJ), relies on the presence of microhomologies within the resected strands of ssDNA, allowing the base pairing of the ssDNA molecules. Subsequently, the missing stretches are filled in by DNA polymerases and ligated. Moreover, an additional pathway with similarities to alt-EJ, known as single-strand annealing (SSA), is driven by extensive resection (>1 × 105 bp) and Rad52-mediated homology search between homologous repeats (Ceccaldi et al., 2016Ceccaldi R. Rondinelli B. D’Andrea A.D. Repair Pathway Choices and Consequences at the Double-Strand Break.Trends Cell Biol. 2016; 26: 52-64Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). This pathway is very mutagenic, as it has been shown to generate deletions of hundreds of kilobases. How these pathways relate to one another is not completely understood. Obviously, these processes are highly toxic for a number of reasons.The use of homology present at both sides of the break will result in the skipping of nucleotides during ligation, producing deletions (Ceccaldi et al., 2016Ceccaldi R. Rondinelli B. D’Andrea A.D. Repair Pathway Choices and Consequences at the Double-Strand Break.Trends Cell Biol. 2016; 26: 52-64Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). In addition, POLQ, the preferred polymerase for MMEJ, has the ability to perform template-independent strand synthesis, resulting in the insertion of random sequences (Kent et al., 2016Kent T. Mateos-Gomez P.A. Sfeir A. Pomerantz R.T. Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining.eLife. 2016; 5: 1-25Crossref Scopus (23) Google Scholar). In contrast, SSA requires long stretches of uninterrupted homology, which is present in repetitive elements such as Alu or alpha satellite repeats (Bhargava et al., 2016Bhargava R. Onyango D.O. Stark J.M. Regulation of Single-Strand Annealing and its Role in Genome Maintenance.Trends Genet. 2016; 32: 566-575Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Recombination between alpha satellite repeats may dramatically reduce centromere function and identity, resulting in chromosome segregation errors, which cause severe toxicity (Figure 3) (Barra and Fachinetti, 2018Barra V. Fachinetti D. The dark side of centromeres: types, causes and consequences of structural abnormalities implicating centromeric DNA.Nat. Commun. 2018; 9: 4340Crossref PubMed Scopus (6) Google Scholar, Tsouroula et al., 2016Tsouroula K. Furst A. Rogier M. Heyer V. Maglott-Roth A. Ferrand A. Reina-San-Martin B. Soutoglou E. Temporal and Spatial Uncoupling of DNA Double Strand Break Repair Pathways within Mammalian Heterochromatin.Mol. Cell. 2016; 63: 293-305Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In the case of Alu repeats, unequal recombination by SSA has been shown to cause multiple syndromes and promotes tumorigenesis (Deininger and Batzer, 1999Deininger P.L. Batzer M.A. Alu repeats and human disease.Mol. Genet. Metab. 1999; 67: 183-193Crossref PubMed Scopus (643) Google Scholar), both of which are due to gene disruption. These properties render SSA a highly mutagenic and detrimental mode of DNA repair. Nonetheless, SSA activity on DSBs in the non-coding genome could be a useful backup repair pathway when other repair pathways are unavailable. Thus, the outcome of any form of end-resection-dependent DNA repair other than HR can be toxic to cell viability due to their highly mutagenic nature. The decision between HR and c-NHEJ is tightly regulated by cell-cycle-dependent phosphorylation and ubiquitination of DNA repair proteins, affecting events that occur following Mre11-dependent break processing (Hustedt and Durocher, 2016Hustedt N. Durocher D. The control of DNA repair by the cell cycle.Nat. Cell Biol. 2016; 19: 1-9Crossref PubMed Scopus (122) Google Scholar) (Figure 2). The repression of c-NHEJ at the lesion is regulated by BRCA1, where it blocks 53BP1 activity (Hustedt and Durocher, 2016Hustedt N. Durocher D. The control of DNA repair by the cell cycle.Nat. Cell Biol. 2016; 19: 1-9Crossref PubMed Scopus (122) Google Scholar, Setiaputra and Durocher, 2019Setiaputra D. Durocher D. Shieldin - the protector of DNA ends.EMBO Rep. 2019; 20: e47560Crossref PubMed Scopus (0) Google Scholar). Conversely, recruitment of the Shieldin complex and 53BP1 prevents BRCA1-driven HR at the break site (Setiaputra and Durocher, 2019Setiaputra D. Durocher D. Shieldin - the protector of DNA ends.EMBO Rep. 2019; 20: e47560Crossref PubMed Scopus (0) Google Scholar). In this manner, both HR and c-NHEJ compete for repair of the same breaks in the S and G2 phases of the cell cycle. There is little evidence to suggest that altering DNA repair pathway choices influences cellular toxicity following break formation. Nonetheless, the loss of several key factors involved in repair pathway choice (e.g., 53BP1, RIF1, REV7, the Shieldin complex) results in mildly increased radiosensitivity (Setiaputra and Durocher, 2019Setiaputra D. Durocher D. Shieldin - the protector of DNA ends.EMBO Rep. 2019; 20: e47560Crossref PubMed Scopus (0) Google Scholar), indicating that the local control of DNA repair pathway usage may influence break-induced toxicity (van den Berg et al., 2019van den Berg J. Joosten S.E.P. Kim Y. Manjón A.G. Krenning L. Koob L. Feringa F.M. Klompmaker R. van den Broek B. Jalink K. et al.DNA end-resection in highly accessible chromatin produces a toxic break.bioRxiv. 2019; https://doi.org/10.1101/691857Crossref Google Scholar). Often overlooked in DNA repair biology are the biochemical properties of the free DNA ends. Free DNA ends exist as a variety of structures, owing to the different causes for a DSB (e.g., exogenous nucleases, chemotherapeutic agents, radiotherapy) (Strande et al., 2012Strande N.T. Waters C.A. Ramsden D.A. Resolution of complex ends by Nonhomologous end joining - better to be lucky than good?.Genome Integr. 2012; 3: 10Crossref PubMed Scopus (7) Google Scholar, Tubbs and Nussenzweig, 2017Tubbs A. Nussenzweig A. Endogenous DNA Damage as a Source of Genomic Instability in Cancer.Cell. 2017; 168: 644-656Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The simplest structure is blunt DSBs, and these are often referred to as “clean” breaks. These structures are preferred by c-NHEJ and will result in the rapid removal of these lesions, and therefore their toxicity is limited. Nonetheless, most chemotherapeutic drugs and radiotherapy as well as cellular processes (e.g., replication stress) do not generate this type of DSB (Strande et al., 2012Strande N.T. Waters C.A. Ramsden D.A. Resolution of complex ends by Nonhomologous end joining - better to be lucky than good?.Genome Integr. 2012; 3: 10Crossref PubMed Scopus (7) Google Scholar, Tubbs and Nussenzweig, 2017Tubbs A. Nussenzweig A. Endogenous DNA Damage as a Source of Genomic Instability in Cancer.Cell. 2017; 168: 644-656Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), so one may argue whether they are a representative model to study the response to DSBs. Most DNA breaks induced by radio- and chemotherapy contain short ssDNA overhangs with modified termini. There is a great diversity in the irradiation-induced DNA termini modifications (Strande et al., 2012Strande N.T. Waters C.A. Ramsden D.A. Resolution of complex ends by Nonhomologous end joining - better to be lucky than good?.Genome Integr. 2012; 3: 10Crossref PubMed Scopus (7) Google Scholar). These terminal modifications require enzymatic processing to form a substrate for DNA ligases. However, it is poorly understood how these different modifications control the speed of DNA repair and contribute to toxicity (Figure 3). Nonetheless, there are some indications that these modifications influence DNA repair speed. For instance, the presence of a 3′-phosphoglycolate (3′-PG), present at ∼50% of DSBs (Henner et al., 1983Henner W.D. Grunberg S.M. Haseltine W.A. Enzyme action at 3′ termini of ionizing radiation-induced DNA strand breaks.J. Biol. Chem. 1983; 258: 15198-15205Abstract Full Text PDF PubMed Google Scholar), requires processing by the Artemis nuclease (Povirk et al., 2007Povirk L.F. Zhou T. Zhou R. Cowan M.J. Yannone S.M. Processing of 3′-phosphoglycolate-terminated DNA double strand breaks by Artemis nuclease.J. Biol. Chem. 2007; 282: 3547-3558Crossref PubMed Scopus (0) Google Scholar). Artemis-dependent NHEJ has been described to be s
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