Replication-Coupled DNA Repair
2019; Elsevier BV; Volume: 74; Issue: 5 Linguagem: Inglês
10.1016/j.molcel.2019.04.027
ISSN1097-4164
Autores Tópico(s)Cancer-related Molecular Pathways
ResumoThe replisome quickly and accurately copies billions of DNA bases each cell division cycle. However, it can make errors, especially when the template DNA is damaged. In these cases, replication-coupled repair mechanisms remove the mistake or repair the template lesions to ensure high fidelity and complete copying of the genome. Failures in these genome maintenance activities generate mutations, rearrangements, and chromosome segregation problems that cause many human diseases. In this review, I provide a broad overview of replication-coupled repair pathways, explaining how they fix polymerase mistakes, respond to template damage that acts as obstacles to the replisome, deal with broken forks, and impact human health and disease. The replisome quickly and accurately copies billions of DNA bases each cell division cycle. However, it can make errors, especially when the template DNA is damaged. In these cases, replication-coupled repair mechanisms remove the mistake or repair the template lesions to ensure high fidelity and complete copying of the genome. Failures in these genome maintenance activities generate mutations, rearrangements, and chromosome segregation problems that cause many human diseases. In this review, I provide a broad overview of replication-coupled repair pathways, explaining how they fix polymerase mistakes, respond to template damage that acts as obstacles to the replisome, deal with broken forks, and impact human health and disease. Spontaneous chemical changes and DNA lesions from endogenous and environmental sources are ubiquitous threats to the information stored in the billions of DNA bases in each human cell. These challenges are acutely problematic during DNA replication and compounded by other forms of replication stress, such as conflicts with transcriptional machineries. Replication-coupled repair, defined as mechanisms that process damaged DNA in coordination with the replisome, works to overcome these challenges and maintain genome stability. Multiple pathways that operate in overlapping layers of repair are engaged based on the type, location, and context of the problem. They include repair activities that remove misincorporation errors; mechanisms to overcome DNA polymerase blocking lesions, like base damage; pathways to deal with replicative helicase-blocking obstacles, including interstrand crosslinks (ICLs) and DNA-protein crosslinks (DPCs); and double-strand break (DSB) repair that protects stalled forks from degradation and restarts broken forks (Figure 1). In addition, the replication machinery can bypass some forms of damage and postpone lesion removal until after DNA synthesis is complete. These are not rarely used mechanisms but rather essential functions needed every cell division cycle. Copying errors and other replication failures cause the genome changes that underlie many human diseases. Most notably, this genetic instability drives cancer development and generates resistance to therapeutic intervention. However, it also provides a difference between cancer and normal cells that can be targeted by chemotherapeutic agents, synthetic lethal approaches with DNA repair inhibitors, and immunotherapy therapeutics. Here, I survey replication-coupled repair mechanisms, consequences of their inactivation, and important unanswered questions. Replicative polymerases are extremely accurate copying machines with self-correction capabilities. Error rates in vitro are higher than what is observed in cells due to additional layers of mismatch correction (Kunkel and Erie, 2015Kunkel T.A. Erie D.A. Eukaryotic mismatch repair in relation to DNA replication.Annu. Rev. Genet. 2015; 49: 291-313Crossref PubMed Scopus (0) Google Scholar). The priming polymerase Polα is the most error prone, but it also performs the least amount of DNA synthesis, and most of the bases it incorporates are removed during Okazaki fragment maturation. The major lagging strand polymerase, Polδ, has intermediate fidelity, and the leading strand polymerase Polε has the highest fidelity of less than one error per 106 bases. Mutations in POLE and POLD1 that reduce proofreading and increase base misincorporation are frequent in a variety of cancer types. A genetically engineered mouse model of one of these POLE mutations confirmed that it greatly increases mutation burden and causes cancer (Li et al., 2018Li H.D. Cuevas I. Zhang M. Lu C. Alam M.M. Fu Y.X. You M.J. Akbay E.A. Zhang H. Castrillon D.H. Polymerase-mediated ultramutagenesis in mice produces diverse cancers with high mutational load.J. Clin. Invest. 2018; 128: 4179-4191Crossref Scopus (5) Google Scholar). Importantly, polymerase mutations may also make these cancers more amenable to treatment because they correlate with a favorable prognosis. One possible explanation is that the high mutation rate generates neoantigens that improve immune system recognition. Misincorporation errors are corrected primarily by mismatch repair (MMR). The detailed mechanisms of MMR will not be covered here; however, a few points are worth noting. First, MMR is more active on the lagging strand than leading, perhaps because of physical interactions between the MutSα and MutLα MMR proteins with PCNA and the prevalence of nicks in the newly synthesized DNA that can act as strand-discrimination signals to tell the MMR machinery which base is incorrect (St Charles et al., 2015St Charles J.A. Liberti S.E. Williams J.S. Lujan S.A. Kunkel T.A. Quantifying the contributions of base selectivity, proofreading and mismatch repair to nuclear DNA replication in Saccharomyces cerevisiae.DNA Repair (Amst.). 2015; 31: 41-51Crossref PubMed Scopus (0) Google Scholar). This increased MMR action on the lagging strand compensates for the reduced fidelity of Polα and Polδ compared to Polε. Second, the MMR machinery is tethered to the replisome via its interaction with PCNA, so it is positioned to fix misincorporation errors as they are made (Hombauer et al., 2011Hombauer H. Campbell C.S. Smith C.E. Desai A. Kolodner R.D. Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates.Cell. 2011; 147: 1040-1053Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, Kleczkowska et al., 2001Kleczkowska H.E. Marra G. Lettieri T. Jiricny J. hMSH3 and hMSH6 interact with PCNA and colocalize with it to replication foci.Genes Dev. 2001; 15: 724-736Crossref PubMed Scopus (179) Google Scholar). PCNA-mediated tethering of repair mechanisms to the replication fork is a common theme in replication-associated repair as exemplified by the large number of repair proteins in the PCNA interactome (Srivastava et al., 2018Srivastava M. Chen Z. Zhang H. Tang M. Wang C. Jung S.Y. Chen J. Replisome dynamics and their functional relevance upon DNA damage through the PCNA interactome.Cell Rep. 2018; 25: 3869-3883.e4Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Third, interactions of PCNA with MMR proteins also can stimulate and direct MutLα−incisions to the nascent strand, providing strand discrimination (Pluciennik et al., 2010Pluciennik A. Dzantiev L. Iyer R.R. Constantin N. Kadyrov F.A. Modrich P. PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair.Proc. Natl. Acad. Sci. USA. 2010; 107: 16066-16071Crossref PubMed Scopus (137) Google Scholar). Fourth, MMR also repairs strand slippage problems that can yield frameshift mutations. Thus, MMR defects cause microsatellite instability (MSI) due to failures in repairing misalignment problems in homonucleotide repeat sequences. This characteristic mutation pattern is used to diagnose tumors caused by MMR deficiencies. Importantly, the increase in neoantigens caused by MSI makes MMR mutant tumors hypersensitive to treatment with immune checkpoint inhibitors (Le et al., 2017Le D.T. Durham J.N. Smith K.N. Wang H. Bartlett B.R. Aulakh L.K. Lu S. Kemberling H. Wilt C. Luber B.S. et al.Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.Science. 2017; 357: 409-413Crossref PubMed Scopus (825) Google Scholar). In addition to inserting incorrect nucleotides, polymerases can also insert damaged bases. Thus, repair mechanisms operate to sanitize the nucleotide pools. However, these mechanisms do not recognize ribonucleotides, which are incorporated in the growing nascent strands hundreds of times more frequently than incorrect DNA bases (Williams et al., 2016Williams J.S. Lujan S.A. Kunkel T.A. Processing ribonucleotides incorporated during eukaryotic DNA replication.Nat. Rev. Mol. Cell Biol. 2016; 17: 350-363Crossref PubMed Scopus (46) Google Scholar). The primary mechanism to remove ribonucleotides is ribonucleotide excision repair (RER). RNaseH2 recognizes the ribose sugar in duplex DNA and cuts the phosphate backbone immediately 5′ to the ribonucleotide. Polδ then performs strand displacement synthesis and the flap endonuclease FEN1 removes the short single-stranded DNA (ssDNA) fragment containing the ribonucleotide. Finally, DNA ligase seals the nick. RER is coupled to DNA replication because the RNaseH2B subunit contains a PCNA-interacting protein (PIP) box that allows it to interact with PCNA (Bubeck et al., 2011Bubeck D. Reijns M.A. Graham S.C. Astell K.R. Jones E.Y. Jackson A.P. PCNA directs type 2 RNase H activity on DNA replication and repair substrates.Nucleic Acids Res. 2011; 39: 3652-3666Crossref PubMed Scopus (60) Google Scholar). Thus, like MMR proteins, RNaseH2 co-localizes with sites of DNA replication. Both MMR and RNaseH2 have also been seen to be part of the replication fork proteome by the iPOND (isolation of proteins on nascent DNA) method that purifies and quantitates the proteins in the replisome and associated with the newly synthesized DNA (Dungrawala et al., 2015Dungrawala H. Rose K.L. Bhat K.P. Mohni K.N. Glick G.G. Couch F.B. Cortez D. The replication checkpoint prevents two types of fork collapse without regulating replisome stability.Mol. Cell. 2015; 59: 998-1010Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). RER inactivation is extremely rare in cancer, but RNaseH2 mutations do cause Aicardi-Goutiéres syndrome (Crow et al., 2006Crow Y.J. Leitch A. Hayward B.E. Garner A. Parmar R. Griffith E. Ali M. Semple C. Aicardi J. Babul-Hirji R. et al.Mutations in genes encoding ribonuclease H2 subunits cause Aicardi-Goutières syndrome and mimic congenital viral brain infection.Nat. Genet. 2006; 38: 910-916Crossref PubMed Scopus (392) Google Scholar). When RNaseH2 is inactivated, TOPO1 can incise the backbone and promote a backup repair pathway (Sekiguchi and Shuman, 1997Sekiguchi J. Shuman S. Site-specific ribonuclease activity of eukaryotic DNA topoisomerase I.Mol. Cell. 1997; 1: 89-97Abstract Full Text Full Text PDF PubMed Google Scholar) but with a significant risk of generating deletions that presumably contributes to the etiology of this disease. The presence of alternative repair mechanisms that contribute to viability but at the expense of genome stability is another common theme of replication-coupled repair and forms the foundation of synthetic lethal approaches to cancer therapy. In addition to misincorporation errors, failures in Okazaki fragment maturation can threaten genome stability. Unligated Okazaki fragments activate PARP1 to promote recruitment of single-strand break repair machinery, including XRCC1 and an alternative DNA ligase—LIG3 (Hanzlikova et al., 2018Hanzlikova H. Kalasova I. Demin A.A. Pennicott L.E. Cihlarova Z. Caldecott K.W. The importance of poly(ADP-ribose) polymerase as a sensor of unligated Okazaki fragments during DNA replication.Mol. Cell. 2018; 71: 319-331.e3Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). This function of PARP1 may contribute to the therapeutic effects of PARP inhibitors in cancer. Most base damage will not block the replicative CMG helicase; however, these lesions will often pause polymerases. The most common base lesions that interfere with DNA synthesis include abasic sites, base oxidation, and base methylation. Approximately 10,000–20,000 abasic sites are formed each day in every human cell through spontaneous base loss and glycosylase removal of uracil or damaged bases. Base oxidation, methylation, thymine glycols, and lipid peroxidation products are additional lesions that form at a rate of a total of ∼20,000 per day. Exposure to environmental contaminants can increase this DNA damage burden, and in tissues exposed to sunlight, UV photoproducts are also common lesions. Although most abasic sites and base lesions are repaired by base excision repair (BER) or nucleotide excision repair (NER) in duplex DNA, these repair systems will not always remove the damage prior to the arrival of a DNA replication fork. The CMG helicase is largely insensitive to their presence, but they can be potent blocks to replicative polymerases. The response to a base lesion depends on whether it is on the leading or lagging template strand (Figure 2). Lagging strand lesions may stall Polα-primase or Polδ but are generally not thought to stall the replication fork or continued DNA synthesis because new primers on the lagging strand naturally facilitate bypass of the problem (Figure 2A). Thus, lagging strand lesions will usually generate gaps that can be repaired or filled in by translesion bypass polymerases post-replicatively—a prediction that has been experimentally verified with reconstituted yeast replication proteins (Taylor and Yeeles, 2018Taylor M.R.G. Yeeles J.T.P. The initial response of a eukaryotic replisome to DNA damage.Mol. Cell. 2018; 70: 1067-1080.e12Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). As yet, verification of this lesion-skipping model in vertebrate cells has not been possible because of the difficulty of directing damage specifically to the lagging strand template. Lesions on the leading strand template would be expected to be more persistent blocks to synthesis because Polα-primase is much less capable of making a new primer on the leading strand (Taylor and Yeeles, 2018Taylor M.R.G. Yeeles J.T.P. The initial response of a eukaryotic replisome to DNA damage.Mol. Cell. 2018; 70: 1067-1080.e12Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Thus, leading strand lesions will often cause the CMG helicase and DNA synthesis to become functionally uncoupled (Figure 2B). The amount of uncoupling in a human cell is not known, and by itself, it may not be particularly unusual or problematic. In E. coli, there is surprisingly little coordination between leading and lagging strand synthesis with or without DNA damage (Graham et al., 2017Graham J.E. Marians K.J. Kowalczykowski S.C. Independent and stochastic action of DNA polymerases in the replisome.Cell. 2017; 169: 1201-1213.e17Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Furthermore, helicase unwinding is reduced ∼80% when a polymerase pauses. This so-called “dead man’s switch” prevents the helicase from running too far ahead of DNA synthesis (Marians, 2018Marians K.J. Lesion bypass and the reactivation of stalled replication forks.Annu. Rev. Biochem. 2018; 87: 217-238Crossref Scopus (13) Google Scholar). Whether the same regulation happens in eukaryotic cells is unknown, although there is evidence from replicating DNA in Xenopus extracts that uncoupling between helicase and polymerase slows the helicase (Sparks et al., 2019Sparks J.L. Chistol G. Gao A.O. Räschle M. Larsen N.B. Mann M. Duxin J.P. Walter J.C. The CMG helicase bypasses DNA-protein cross-links to facilitate their repair.Cell. 2019; 176: 167-181.e21Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Nonetheless, uncoupling in the Xenopus system can be quite extensive, leading to unwinding of kilobases of DNA (Byun et al., 2005Byun T.S. Pacek M. Yee M.C. Walter J.C. Cimprich K.A. Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.Genes Dev. 2005; 19: 1040-1052Crossref PubMed Scopus (468) Google Scholar). Such large amounts of ssDNA have not been observed in vertebrate cells, suggesting the Xenopus egg extracts may not reflect a typical cellular response, but even small amounts of uncoupling can generate a platform for recruitment of ssDNA binding proteins that initiate ATR-dependent replication stress signaling or recruit other repair proteins (Figure 2B). Eventually, a new primer is made on the leading strand template, allowing DNA synthesis to resume, leaving an ssDNA gap on the leading strand. Repriming in E. coli happens within minutes and does not require a specialized primase (Yeeles and Marians, 2011Yeeles J.T. Marians K.J. The Escherichia coli replisome is inherently DNA damage tolerant.Science. 2011; 334: 235-238Crossref PubMed Scopus (78) Google Scholar). Thus, both lagging and leading strand damage is skipped by the bacterial replisome, although with different kinetics. The eukaryotic replisome has some ability to skip leading-strand lesions but appears to preferentially use a specialized primase called PrimPol (Bianchi et al., 2013Bianchi J. Rudd S.G. Jozwiakowski S.K. Bailey L.J. Soura V. Taylor E. Stevanovic I. Green A.J. Stracker T.H. Lindsay H.D. Doherty A.J. PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication.Mol. Cell. 2013; 52: 566-573Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, García-Gómez et al., 2013García-Gómez S. Reyes A. Martínez-Jiménez M.I. Chocrón E.S. Mourón S. Terrados G. Powell C. Salido E. Méndez J. Holt I.J. Blanco L. PrimPol, an archaic primase/polymerase operating in human cells.Mol. 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Alternatively, a template-switching mechanism could utilize the information from the undamaged sister chromatid as a template for synthesis in an error-free manner. If most DNA lesions generate gaps behind the replication fork, this would explain how the ATR replication checkpoint is readily activated because TOPBP1-dependent activation requires a 5′ DNA end adjacent to ssDNA to be activated by the TOPBP1 (Saldivar et al., 2018Saldivar J.C. Hamperl S. Bocek M.J. Chung M. Bass T.E. Cisneros-Soberanis F. Samejima K. Xie L. Paulson J.R. Earnshaw W.C. et al.An intrinsic S/G2 checkpoint enforced by ATR.Science. 2018; 361: 806-810Crossref PubMed Scopus (5) Google Scholar). In cases where there is no 5′ DNA junction, an alternative ATR activation pathway dependent on the ETAA1 ATR activator may still operate as long as there is a region of replication protein A (RPA)-coated ssDNA, but this pathway appears to be less involved in replication-stress-induced signaling than the TOPBP1 pathway and relatively more important for controlling cell division processes independent of DNA damage (Bass and Cortez, 2019Bass T.E. Cortez D. Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1.J. Cell Biol. 2019; 218: 1235-1249Crossref Scopus (2) Google Scholar, Bass et al., 2016Bass T.E. Luzwick J.W. Kavanaugh G. Carroll C. Dungrawala H. Glick G.G. Feldkamp M.D. Putney R. Chazin W.J. Cortez D. ETAA1 acts at stalled replication forks to maintain genome integrity.Nat. Cell Biol. 2016; 18: 1185-1195Crossref PubMed Google Scholar, Haahr et al., 2016Haahr P. Hoffmann S. Tollenaere M.A. Ho T. Toledo L.I. Mann M. Bekker-Jensen S. Räschle M. Mailand N. Activation of the ATR kinase by the RPA-binding protein ETAA1.Nat. Cell Biol. 2016; 18: 1196-1207Crossref PubMed Google Scholar, Saldivar et al., 2018Saldivar J.C. Hamperl S. Bocek M.J. Chung M. Bass T.E. Cisneros-Soberanis F. Samejima K. Xie L. Paulson J.R. Earnshaw W.C. et al.An intrinsic S/G2 checkpoint enforced by ATR.Science. 2018; 361: 806-810Crossref PubMed Scopus (5) Google Scholar). Activated ATR signals to a large number of pathways to promote repair, fork restart, and delay progression through the cell cycle (Saldivar et al., 2018Saldivar J.C. Hamperl S. Bocek M.J. Chung M. Bass T.E. Cisneros-Soberanis F. Samejima K. Xie L. Paulson J.R. Earnshaw W.C. et al.An intrinsic S/G2 checkpoint enforced by ATR.Science. 2018; 361: 806-810Crossref PubMed Scopus (5) Google Scholar). An alternative mechanism for dealing with fork-stalling lesions is fork reversal, which is catalyzed by fork reversal enzymes, including SMARCAL1, ZRANB3, and HLTF (Bétous et al., 2012Bétous R. 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Fork reversal involves migrating the three-way fork junction backward to displace and anneal the nascent DNA strands to form what is called a chicken foot structure (Figures 2B and 3). Once thought to be rare in eukaryotes because reversed forks were only observed by electron microscopy (EM) in replication checkpoint-deficient budding yeast cells (Sogo et al., 2002Sogo J.M. Lopes M. Foiani M. Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects.Science. 2002; 297: 599-602Crossref PubMed Scopus (589) Google Scholar), recent studies indicate that fork reversal may be quite frequent in vertebrates. In fact, EM analyses of replication forks purified from mammalian cells revealed that ∼25% of the detected forks are reversed in cells treated with agents that induce nucleotide depletion, oxidative base damage, UV photoproducts, topoisomerase cleavage complexes, or DNA crosslinks (Zellweger et al., 2015Zellweger R. Dalcher D. Mutreja K. Berti M. Schmid J.A. Herrador R. Vindigni A. Lopes M. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.J. Cell Biol. 2015; 208: 563-579Crossref PubMed Scopus (182) Google Scholar). This important result suggests that fork reversal is common, even when the fork encounters lesions that should be relatively easy to skip. One caveat to this interpretation is that the EM analysis assumes equivalent frequencies of psoralen-dependent stabilization and subsequent purification of replication intermediates to derive a quantitation of fork-reversal frequency. Furthermore, the method is resource and time intensive, limiting how much data can be acquired. Thus, although the relative levels of fork reversal reported are informative, the field would benefit from development of an independent method to ascertain the relative and absolute percentages of reversed forks. In any case, based on the phenotypes associated with inactivating the fork reversal enzymes SMARCAL1, ZRANB3, or HLTF, fork reversal must be a significant replication stress tolerance mechanism. Inactivating each of these genes individually alters sensitivity to replication stress, changes replication fork progression, and induces genome instability (Bansbach et al., 2009Bansbach C.E. Bétous R. Lovejoy C.A. Glick G.G. Cortez D. The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks.Genes Dev. 2009; 23: 2405-2414Crossref PubMed Scopus (136) Google Scholar, Blastyák et al., 2010Blastyák A. Hajdú I. Unk I. Haracska L. Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA.Mol. Cell. Biol. 2010; 30: 684-693Crossref PubMed Scopus (94) Google Scholar, Ciccia et al., 2009Ciccia A. Bredemeyer A.L. Sowa M.E. Terret M.E. Jallepalli P.V. Harper J.W. Elledge S.J. 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Fork reversal has several potential benefits. First, it may be a way to place a template DNA lesion back into the context of duplex DNA, where excision repair mechanisms can operate. Although intuitively attractive, there are little direct data on whether excision repair is actually coupled to reversal, so experimentally testing this model of replication-coupled repair is a high priority. One possibility is that ZRANB3 directly participates in removal of the DNA lesion because it contains an endonuclease activity in addition to its fork-remodeling function (Weston et al., 2012Weston R. Peeters H. Ahel D. ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response.Genes Dev. 2012; 26: 1558-1572Crossref PubMed Scopus (56) Google Scholar). This endonuclease is unusual in that it depends on both the ATPase motor and DNA substrate recognition domain of ZRANB3, which may ensure that DNA scission is coupled to fork reversal (Badu-Nkansah et al., 2016Badu-Nkansah A. Mason A.C. Eichman B.F. Cortez D. Identification of a substrate recognition domain in the replication stress response protein zinc finger Ran-binding domain-containing protein 3 (ZRANB3).J. Biol. Chem. 2016; 291: 8251-8257Crossref PubMed Scopus (9) Google Scholar, Weston et al., 2012Weston R. Peeters H. Ahel D. ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response.Genes Dev. 2012; 26: 1558-1572Crossref PubMed Scopus (56) Google Scholar). Second, fork reversal could be a mechanism of template switching using the newly synthesized DNA strand as an undamaged template. Third, fork reversal may be a way of sequestering the stalled fork until a converging fork initiated from another origin finishes DNA synthesis of the region. Fourth, reversal is an essential step in some repair mechanisms, such as when two forks converge on an interstrand crosslink (Amunugama et al., 2018Amunugama R. Willcox S. Wu R.A. Abdullah U.B. El-Sagheer A.H. Brown T. McHugh P.J. Griffith J.D. Walter J.C. Replication fork reversal during DNA interstrand crosslink repair requires CMG unloading.Cell Rep. 2018; 23: 3419-3428Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Finally, fork reversal could be an intermediate in a recombination pathway of fork restart. Recombination proteins, including RAD51, BRCA1, and BRCA2, do have critical functions in fork-reversal pathways. RAD51 is needed to generate reversed forks (Zellweger et al., 2015Zellweger R. Dalcher D. Mutreja K. Berti M. Schmid J.A. Herrador R. Vindigni A. Lopes M. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.J. Cell Biol. 2015; 208: 563-579Crossref PubMed Scopus (182) Google Scholar). However, what it does to promote reversal is unknown. One model is that RAD51 binds to the ssDNA on the template strands to do some kind of coordinated annealing reactio
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