DNA lesion identity drives choice of damage tolerance pathway in murine cell chromosomes

DNA-damage tolerance (DDT) via translesion DNA synthesis (TLS) or homology-dependent repair (HDR) functions to bypass DNA lesions encountered during replication, and is critical for maintaining genome stability. Here, we present piggyBlock, a new chromosomal assay that, using piggyBac transposition of DNA containing a known lesion, measures the division of labor between the two DDT pathways. We show that in the absence of DNA damage response, tolerance of the most common sunlight-induced DNA lesion, TT-CPD, is achieved by TLS in mouse embryo fibroblasts. Meanwhile, BP-G, a major smoke-induced DNA lesion, is bypassed primarily by HDR, providing the first evidence for this mechanism being the main tolerance pathway for a biologically important lesion in a mammalian genome. We also show that, far from being a last-resort strategy as it is sometimes portrayed, TLS operates alongside nucleotide excision repair, handling 40% of TT-CPDs in repair-proficient cells. Finally, DDT acts in mouse embryonic stem cells, exhibiting the same pattern—mutagenic TLS included—despite the risk of propagating mutations along all cell lineages. The new method highlights the importance of HDR, and provides an effective tool for studying DDT in mammalian cells.     

RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells

Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major pathways that account for survival after post-replicational DNA damage. TLS functions by filling gaps on a daughter strand that remain after DNA replication caused by damage on the mother strand, while HR can repair gaps and breaks using the intact sister chromatid as a template. The RAD18 gene, which is conserved from lower eukaryotes to vertebrates, is essential for TLS in Saccharomyces cerevisiae. To investigate the role of RAD18, we disrupted RAD18 by gene targeting in the chicken B-lymphocyte line DT40. RAD18(-/-) cells are sensitive to various DNA-damaging agents including ultraviolet light and the cross-linking agent cisplatin, consistent with its role in TLS. Interestingly, elevated sister chromatid exchange, which reflects HR- mediated post-replicational repair, was observed in RAD18(-/-) cells during the cell cycle. Strikingly, double mutants of RAD18 and RAD54, a gene involved in HR, are synthetic lethal, although the single mutant in either gene can proliferate with nearly normal kinetics. These data suggest that RAD18 plays an essential role in maintaining chromosomal DNA in cooperation with the RAD54-dependent DNA repair pathway.     

The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.     

Non‐recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance

DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error‐prone, HR uses the sister chromatid and is mostly error‐free. We report that the HR protein Rad52—but not Rad51 and Rad57—acts in concert with the TLS machinery (Rad6/Rad18‐mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light‐induced ssDNA gaps through a non‐recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS‐ and UV‐induced HR); accordingly, Rad52 is required for efficient DNA damage‐induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage‐induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57‐dependent and ‐independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.     

The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line

The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase. To explore the role of HERC2 and RNF8 in PRR, we examined immunoglobulin diversification in chicken DT40 cells deficient in HERC2 and RNF8. Unexpectedly, the HERC2^−/− and RNF8^−/− cells and HERC2^−/−/RNF8^−/− double mutant cells exhibit a significant reduction in the rate of immunoglobulin (Ig) hypermutation, compared to wild-type cells. Further, the HERC2^−/− and RNF8^−/− mutants exhibit defective maintenance of replication fork progression immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks.     

Involvement of vertebrate polkappa in Rad18-independent postreplication repair of UV damage

DNA damage, which is left unrepaired by excision repair pathways, often blocks replication, leading to lesions such as breaks and gaps on the sister chromatids. These lesions may be processed by either homologous recombination (HR) repair or translesion DNA synthesis (TLS). Vertebrate Polkappa belongs to the DNA polymerase Y family, as do most TLS polymerases. However, the role for Polkappa in vertebrate cells is unclear because of the lack of reverse genetic studies. Here, we generated cells deficient in Polkappa (polkappa cells) from the chicken B lymphocyte line DT40. Although purified Polkappa is unable to bypass ultraviolet (UV) damage, polkappa cells exhibited increased UV sensitivity, and the phenotype was suppressed by expression of human and chicken Polkappa, suggesting that Polkappa is involved in TLS of UV photoproduct. Defects in both Polkappa and Rad18, which regulates TLS in yeast, in DT40 showed an additive effect on UV sensitivity. Interestingly, the level of sister chromatid exchange, which reflects HR-mediated repair, was elevated in normally cycling polkappa cells. This implies functional redundancy between HR and Polkappa in maintaining chromosomal DNA. In conclusion, vertebrate Polkappa is involved in Rad18-independent TLS of UV damage and plays a role in maintaining genomic stability.     

Analysis of strand transfer and template switching mechanisms of DNA gap repair by homologous recombination in Escherichia coli: predominance of strand transfer

Daughter strand gaps formed upon interruption of replication at DNA lesions in Escherichiacoli can be repaired by either translesion DNA synthesis or homologous recombination (HR) repair. Using a plasmid-based assay system that enables discrimination between strand transfer and template switching (information copying) modes of HR gap repair, we found that approximately 80% of strand gaps were repaired by physical strand transfer from the donor, whereas approximately 20% appear to be repaired by template switching. HR gap repair operated on both small and bulky lesions and largely depended on RecA and RecF but not on the RecBCD nuclease. In addition, we found that HR was mildly reduced in cells lacking the RuvABC and RecG proteins involved in resolution of Holliday junctions. These results, obtained for the first time under conditions that detect the two HR gap repair mechanisms, provide in vivo high-resolution molecular evidence for the predominance of the strand transfer mechanism in HR gap repair. A small but significant portion of HR gap repair appears to occur via a template switching mechanism.     

Multiple pathways cooperate to facilitate DNA replication fork progression through alkylated DNA

Eukaryotic genomes are especially vulnerable to DNA damage during the S phase of the cell cycle, when chromosomes must be duplicated. The stability of DNA replication forks is critical to achieve faithful chromosome replication and is severely compromised when forks encounter DNA lesions. To maintain genome integrity, replication forks need to be protected by the S-phase checkpoint and DNA insults must be repaired. Different pathways help to repair or tolerate the lesions in the DNA, but their contribution to the progression of replication forks through damaged DNA is not well known. Here we show in budding yeast that, when the DNA template is damaged with the alkylating agent methyl methanesulfonate (MMS), base excision repair, homologous recombination and DNA damage tolerance pathways, together with a functional S-phase checkpoint, are essential for the efficient progression of DNA replication forks and the maintenance of cell survival. In the absence of base excision repair, replication forks stall reversibly in cells exposed to MMS. This repair reaction is necessary to eliminate the lesions that impede fork progression and has to be coordinated with recombination and damage tolerance activities to avoid fork collapse and allow forks to resume and complete chromosome replication.     

Mechanism of DNA damage tolerance

DNA damage may compromise genome integrity and lead to cell death. Cells have evolved a variety of processes to respond to DNA damage including damage repair and tolerance mechanisms, as well as damage checkpoints. The DNA damage tolerance(DDT) pathway promotes the bypass of single-stranded DNA lesions encountered by DNA polymerases during DNA replication. This prevents the stalling of DNA replication. Two mechanistically distinct DDT branches have been characterized. One is translesion synthesis(TLS) in which a replicative DNA polymerase is temporarily replaced by a specialized TLS polymerase that has the ability to replicate across DNA lesions. TLS is mechanistically simple and straightforward, but it is intrinsically error-prone. The other is the error-free template switching(TS) mechanism in which the stalled nascent strand switches from the damaged template to the undamaged newly synthesized sister strand for extension past the lesion. Error-free TS is a complex but preferable process for bypassing DNA lesions. However, our current understanding of this pathway is sketchy. An increasing number of factors are being found to participate or regulate this important mechanism, which is the focus of this editorial.     

DNA bending facilitates the error‐free DNA damage tolerance pathway and upholds genome integrity

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination‐mediated (error‐free) and translesion synthesis‐mediated (error‐prone) DNA damage tolerance pathways. Crucial for error‐free DNA damage tolerance is template switching, which depends on the formation and resolution of damage‐bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication‐associated lesions into the error‐free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C‐terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication‐associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.     

Bacterial DNA repair genes and their eukaryotic homologues: 5. The role of recombination in DNA repair and genome stability

Recombinational repair is a well conserved DNA repair mechanism present in all living organisms. Repair by homologous recombination is generally accurate as it uses undamaged homologous DNA molecule as a repair template. In Escherichia coli homologous recombination repairs both the double-strand breaks and single-strand gaps in DNA. DNA double-strand breaks (DSB) can be induced upon exposure to exogenous sources such as ionizing radiation or endogenous DNA-damaging agents including reactive oxygen species (ROS) as well as during natural biological processes like conjugation. However, the bulk of double strand breaks are formed during replication fork collapse encountering an unrepaired single strand gap in DNA. Under such circumstances DNA replication on the damaged template can be resumed only if supported by homologous recombination. This functional cooperation of homologous recombination with replication machinery enables successful completion of genome duplication and faithful transmission of genetic material to a daughter cell. In eukaryotes, homologous recombination is also involved in essential biological processes such as preservation of genome integrity, DNA damage checkpoint activation, DNA damage repair, DNA replication, mating type switching, transposition, immune system development and meiosis. When unregulated, recombination can lead to genome instability and carcinogenesis.     

Coordination of DNA replication and recombination activities in the maintenance of genome stability

Across the evolutionary spectrum, living organisms depend on high-fidelity DNA replication and recombination mechanisms to maintain genome stability and thus to avoid mutation and disease. The repair of severe lesions in the DNA such as double-strand breaks or stalled replication forks requires the coordinated activities of both the homologous recombination (HR) and DNA replication machineries. Growing evidence indicates that so-called "accessory proteins" in both systems are essential for the effective coupling of recombination to replication which is necessary to restore genome integrity following severe DNA damage. In this article we review the major processes of homology-directed DNA repair (HDR), including the double Holliday Junction (dHJ), synthesis-dependent strand annealing (SDSA), break-induced replication (BIR), and error-free lesion bypass pathways. Each of these pathways involves the coupling of a HR event to DNA synthesis. We highlight two major classes of accessory proteins in recombination and replication that facilitate HDR: Recombination mediator proteins exemplified by T4 UvsY, Saccharomyces cerevisiae Rad52, and human BRCA2; and DNA helicases/translocases exemplified by T4 Gp41/Gp59, E. coli DnaB and PriA, and eukaryotic Mcm2-7, Rad54, and Mph1. We illustrate how these factors help to direct the flow of DNA and protein-DNA intermediates on the pathway from a double-strand break or stalled replication fork to a high-fidelity recombination-dependent replication apparatus that can accurately repair the damage.     

Regulation of tolerance to DNA alkylating damage by Dot1 and Rad53 in Saccharomyces cerevisiae

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous factors that produce genome injuries and interfere with DNA replication. DNA integrity checkpoints coordinate this response by slowing cell cycle progression to provide time for the cell to repair the damage, stabilizing replication forks and stimulating DNA repair to restore the original DNA sequence and structure. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage. TLS allows replication to continue without removing the damage, but results in a higher frequency of mutagenesis. Here, we investigate the functional contribution of the Dot1 histone methyltransferase and the Rad53 checkpoint kinase to TLS regulation in Saccharomyces cerevisiae. We demonstrate that the Dot1-dependent status of H3K79 methylation modulates the resistance to the alkylating agent MMS, which depends on PCNA ubiquitylation at lysine 164. Strikingkly, either the absence of DOT1, which prevents full activation of Rad53, or the expression of an HA-tagged version of RAD53, which produces low amounts of the kinase, confer increased MMS resistance. However, the dot1Δ rad53-HA double mutant is hypersensitive to MMS and shows barely detectable amounts of activated kinase. Furthermore, moderate overexpression of RAD53 partially suppresses the MMS resistance of dot1Δ. In addition, we show that MMS-treated dot1Δ and rad53-HA cells display increased number of chromosome-associated Rev1 foci. We propose that threshold levels of Rad53 activity exquisitely modulate the tolerance to alkylating damage at least by controlling the abundance of the key TLS factor Rev1 bound to chromatin.     

A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity

Accurate DNA double-strand break repair through homologous recombination is essential for preserving genome integrity. Disruption of the gene encoding RAD51, the protein that catalyzes DNA strand exchange during homologous recombination, results in lethality of mammalian cells. Proteins required for homologous recombination, also play an important role during DNA replication. To explore the role of RAD51 in DNA replication and DSB repair, we used a knock-in strategy to express a carboxy-terminal fusion of green fluorescent protein to mouse RAD51 (mRAD51-GFP) in mouse embryonic stem cells. Compared to wild-type cells, heterozygous mRad51^+/wt-GFP embryonic stem cells showed increased sensitivity to DNA damage induced by ionizing radiation and mitomycin C. Moreover, gene targeting was found to be severely impaired in mRad51^+/wt-GFP embryonic stem cells. Furthermore, we found that mRAD51-GFP foci were not stably associated with chromatin. From these experiments we conclude that this mRad51-GFP allele is an antimorphic allele. When this allele is present in a heterozygous condition over wild-type mRad51, embryonic stem cells are proficient in DNA replication but display defects in homologous recombination and DNA damage repair.     

DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity

Translesion DNA synthesis (TLS) employs low-fidelity DNA polymerases to bypass replication-blocking lesions, and being associated with chromosomal replication was presumed to occur in the S phase of the cell cycle. Using immunostaining with anti-replication protein A antibodies, we show that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persist into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase ζ and Rev1. Analysis of TLS using a high-resolution gapped-plasmid assay system in cell populations enriched by centrifugal elutriation for specific cell cycle phases showed that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions can lag behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. Such a mechanism may function to maintain efficient replication, which can progress despite the presence of DNA lesions, with TLS lagging behind and patching regions of discontinuity.     

Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation.     

Translesion DNA synthesis in the context of cancer research

During cell division, replication of the genomic DNA is performed by high-fidelity DNA polymerases but these error-free enzymes can not synthesize across damaged DNA. Specialized DNA polymerases, so called DNA translesion synthesis polymerases (TLS polymerases), can replicate damaged DNA thereby avoiding replication fork breakdown and subsequent chromosomal instability. We focus on the involvement of mammalian TLS polymerases in DNA damage tolerance mechanisms. In detail, we review the discovery of TLS polymerases and describe the molecular features of all the mammalian TLS polymerases identified so far. We give a short overview of the mechanisms that regulate the selectivity and activity of TLS polymerases. In addition, we summarize the current knowledge how different types of DNA damage, relevant either for the induction or treatment of cancer, are bypassed by TLS polymerases. Finally, we elucidate the relevance of TLS polymerases in the context of cancer therapy.     

The involvement of Srs2 in post-replication repair and homologous recombination in fission yeast

Homologous recombination is important for the repair of double-strand breaks and daughter strand gaps, and also helps restart stalled and collapsed replication forks. However, sometimes recombination is inappropriate and can have deleterious consequences. To temper recombination, cells have employed DNA helicases that unwind joint DNA molecules and/or dissociate recombinases from DNA. Budding yeast Srs2 is one such helicase. It can act by dissociating Rad51 nucleoprotein filaments, and is required for channelling DNA lesions to the post-replication repair (PRR) pathway. Here we have investigated the role of Srs2 in controlling recombination in fission yeast. Similar to budding yeast, deletion of fission yeast srs2 results in hypersensitivity to a range of DNA damaging agents, rhp51-dependent hyper-recombination and synthetic sickness when combined with rqh1^– that is suppressed by deleting rhp51, rhp55 or rhp57. Epistasis analysis indicates that Srs2 and the structure-specific endonuclease Mus81–Eme1 function in a sub-pathway of PRR for the tolerance/repair of UV-induced damage. However, unlike in Saccharomyces cerevisiae, Srs2 is not required for channelling lesions to the PRR pathway in Schizosaccharomyces pombe. In addition to acting as an antirecombinase, we also show that Srs2 can aid the recombinational repair of camptothecin-induced collapsed replication forks, independently of PRR.     

DNA2 and EXO1 in replication-coupled,homology-directed repair and in the interplay between HDR and the FA/BRCA network

During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.