SnapShot: DNA repair pathways.

The genetic information stored in DNA is under continuous threat by endogenous and environmental sources of DNA damage. Cells have evolved multiple DNA repair pathways that function in overlapping manners, with principles shared across species. Here, we depict the main DNA repair pathways cells rely on, with the primary lesions they are tackling, along with key players and main DNA transactions. To view this SnapShot, open or download the PDF.

SnapShot: Tolerating replication stress.

Completion of DNA replication relies on the ability of replication forks to traverse various types of DNA damage, actively transcribed regions, and structured DNA. The mechanisms enabling these processes are here referred to as DNA damage tolerance pathways. Here, we depict the stalled DNA replication fork structures with main DNA transactions and key factors contributing to the bypass of such blocks, replication restart, and completion. To view this SnapShot, open or download the PDF.

Parental histone deposition on the replicated strands promotes error-free DNA damage tolerance and regulates drug resistance.

Ctf4 is a conserved replisome component with multiple roles in DNA metabolism. To investigate connections between Ctf4-mediated processes involved in drug resistance, we conducted a suppressor screen of ctf4Δ sensitivity to the methylating agent MMS. We uncovered that mutations in Dpb3 and Dpb4 components of polymerase ε result in the development of drug resistance in ctf4Δ via their histone-binding function. Alleviated sensitivity to MMS of the double mutants was not associated with rescue of ctf4Δ defects in sister chromatid cohesion, replication fork architecture, or template switching, which ensures error-free replication in the presence of genotoxic stress. Strikingly, the improved viability depended on translesion synthesis (TLS) polymerase-mediated mutagenesis, which was drastically increased in ctf4 dpb3 double mutants. Importantly, mutations in Mcm2-Ctf4-Polα and Dpb3-Dpb4 axes of parental (H3-H4)2 deposition on lagging and leading strands invariably resulted in reduced error-free DNA damage tolerance through gap filling by template switch recombination. Overall, we uncovered a chromatin-based drug resistance mechanism in which defects in parental histone transfer after replication fork passage impair error-free recombination bypass and lead to up-regulation of TLS-mediated mutagenesis and drug resistance.

Vertebrate CTF18 and DDX11 essential function in cohesion is bypassed by preventing WAPL-mediated cohesin release.

The alternative PCNA loader containing CTF18-DCC1-CTF8 facilitates sister chromatid cohesion (SCC) by poorly defined mechanisms. Here we found that in DT40 cells, CTF18 acts complementarily with the Warsaw breakage syndrome DDX11 helicase in mediating SCC and proliferation. We uncover that the lethality and cohesion defects of ctf18 ddx11 mutants are associated with reduced levels of chromatin-bound cohesin and rescued by depletion of WAPL, a cohesin-removal factor. On the contrary, high levels of ESCO1/2 acetyltransferases that acetylate cohesin to establish SCC do not rescue ctf18 ddx11 phenotypes. Notably, the tight proximity of sister centromeres and increased anaphase bridges characteristic of WAPL-depleted cells are abrogated by loss of both CTF18 and DDX11 The results reveal that vertebrate CTF18 and DDX11 collaborate to provide sufficient amounts of chromatin-loaded cohesin available for SCC generation in the presence of WAPL-mediated cohesin-unloading activity. This process modulates chromosome structure and is essential for cellular proliferation in vertebrates.

SUMO-Chain-Regulated Proteasomal Degradation Timing Exemplified in DNA Replication Initiation.

Similar to ubiquitin, SUMO forms chains, but the identity of SUMO-chain-modified factors and the purpose of this modification remain largely unknown. Here, we identify the budding yeast SUMO protease Ulp2, able to disassemble SUMO chains, as a DDK interactor enriched at replication origins that promotes DNA replication initiation. Replication-engaged DDK is SUMOylated on chromatin, becoming a degradation-prone substrate when Ulp2 no longer protects it against SUMO chain assembly. Specifically, SUMO chains channel DDK for SUMO-targeted ubiquitin ligase Slx5/Slx8-mediated and Cdc48 segregase-assisted proteasomal degradation. Importantly, the SUMOylation-defective ddk-KR mutant rescues inefficient replication onset and MCM activation in cells lacking Ulp2, suggesting that SUMO chains time DDK degradation. Using two unbiased proteomic approaches, we further identify subunits of the MCM helicase and other factors as SUMO-chain-modified degradation-prone substrates of Ulp2 and Slx5/Slx8. We thus propose SUMO-chain/Ulp2-protease-regulated proteasomal degradation as a mechanism that times the availability of functionally engaged SUMO-modified protein pools during replication and beyond.

Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates.

Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.

FANCJ DNA helicase is recruited to the replisome by AND-1 to ensure genome stability.

FANCJ, a DNA helicase linked to Fanconi anemia and frequently mutated in cancers, counteracts replication stress by dismantling unconventional DNA secondary structures (such as G-quadruplexes) that occur at the DNA replication fork in certain sequence contexts. However, how FANCJ is recruited to the replisome is unknown. Here, we report that FANCJ directly binds to AND-1 (the vertebrate ortholog of budding yeast Ctf4), a homo-trimeric protein adaptor that connects the CDC45/MCM2-7/GINS replicative DNA helicase with DNA polymerase α and several other factors at DNA replication forks. The interaction between FANCJ and AND-1 requires the integrity of an evolutionarily conserved Ctf4-interacting protein (CIP) box located between the FANCJ helicase motifs IV and V. Disruption of the CIP box significantly reduces FANCJ association with the replisome, causing enhanced DNA damage, decreased replication fork recovery and fork asymmetry in cells unchallenged or treated with Pyridostatin, a G-quadruplex-binder, or Mitomycin C, a DNA inter-strand cross-linking agent. Cancer-relevant FANCJ CIP box variants display reduced AND-1-binding and enhanced DNA damage, a finding that suggests their potential role in cancer predisposition.

ESCO1/2's roles in chromosome structure and interphase chromatin organization.

ESCO1/2 acetyltransferases mediating SMC3 acetylation and sister chromatid cohesion (SCC) are differentially required for genome integrity and development. Here we established chicken DT40 cell lines with mutations in ESCO1/2, SMC3 acetylation, and the cohesin remover WAPL. Both ESCO1 and ESCO2 promoted SCC, while ESCO2 was additionally and specifically required for proliferation and centromere integrity. ESCO1 overexpression fully suppressed the slow proliferation and centromeric separation phenotypes of esco2 cells but only partly suppressed its chromosome arm SCC defects. Concomitant inactivation of ESCO1 and ESCO2 caused lethality owing to compromised mitotic chromosome segregation. Neither wapl nor acetyl-mimicking smc3-QQ mutations rescued esco1 esco2 lethality. Notably, esco1 esco2 wapl conditional mutants showed very severe proliferation defects associated with catastrophic mitoses and also abnormal interphase chromatin organization patterns. The results indicate that cohesion establishment by vertebrate ESCO1/2 is linked to interphase chromatin architecture formation, a newly identified function of cohesin acetyltransferases that is both fundamentally and medically relevant.

Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication.

Regions of the genome with the potential to form secondary DNA structures pose a frequent and significant impediment to DNA replication and must be actively managed in order to preserve genetic and epigenetic integrity. How the replisome detects and responds to secondary structures is poorly understood. Here, we show that a core component of the fork protection complex in the eukaryotic replisome, Timeless, harbours in its C-terminal region a previously unappreciated DNA-binding domain that exhibits specific binding to G-quadruplex (G4) DNA structures. We show that this domain contributes to maintaining processive replication through G4-forming sequences, and exhibits partial redundancy with an adjacent PARP-binding domain. Further, this function of Timeless requires interaction with and activity of the helicase DDX11. Loss of both Timeless and DDX11 causes epigenetic instability at G4-forming sequences and DNA damage. Our findings indicate that Timeless contributes to the ability of the replisome to sense replication-hindering G4 formation and ensures the prompt resolution of these structures by DDX11 to maintain processive DNA synthesis.

DDX11 loss causes replication stress and pharmacologically exploitable DNA repair defects.

DDX11 encodes an iron-sulfur cluster DNA helicase required for development, mutated, and overexpressed in cancers. Here, we show that loss of DDX11 causes replication stress and sensitizes cancer cells to DNA damaging agents, including poly ADP ribose polymerase (PARP) inhibitors and platinum drugs. We find that DDX11 helicase activity prevents chemotherapy drug hypersensitivity and accumulation of DNA damage. Mechanistically, DDX11 acts downstream of 53BP1 to mediate homology-directed repair and RAD51 focus formation in manners nonredundant with BRCA1 and BRCA2. As a result, DDX11 down-regulation aggravates the chemotherapeutic sensitivity of BRCA1/2-mutated cancers and resensitizes chemotherapy drug-resistant BRCA1/2-mutated cancer cells that regained homologous recombination proficiency. The results further indicate that DDX11 facilitates recombination repair by assisting double strand break resection and the loading of both RPA and RAD51 on single-stranded DNA substrates. We propose DDX11 as a potential target in cancers by creating pharmacologically exploitable DNA repair vulnerabilities.

Mutation in senataxin alters the mechanism of R-loop resolution in amyotrophic lateral sclerosis 4.

Mutation in the senataxin (SETX) gene causes an autosomal dominant neuromuscular disorder, amyotrophic lateral sclerosis 4 (ALS4), characterized by degeneration of motor neurons, muscle weakness and atrophy. SETX is an RNA-DNA helicase that mediates resolution of co-transcriptional RNA:DNA hybrids (R-loops). The process of R-loop resolution is essential for the normal functioning of cells, including neurons. The molecular basis of ALS4 pathogenesis and the mechanism of R-loop resolution are unclear. We report that the zinc finger protein ZPR1 binds to RNA:DNA hybrids, recruits SETX onto R-loops and is critical for R-loop resolution. ZPR1 deficiency disrupts the integrity of R-loop resolution complexes containing SETX and causes increased R-loop accumulation throughout gene transcription. We uncover that SETX is a downstream target of ZPR1 and that overexpression of ZPR1 can rescue R-loop resolution complexe assembly in SETX-deficient cells but not vice versa. To uncover the mechanism of R-loop resolution, we examined the function of SETX-ZPR1 complexes using two genetic motor neuron disease models with altered R-loop resolution. Notably, chronic low levels of SETX-ZPR1 complexes onto R-loops result in a decrease of R-loop resolution activity causing an increase in R-loop levels in spinal muscular atrophy. ZPR1 overexpression increases recruitment of SETX onto R-loops, decreases R-loops and rescues the spinal muscular atrophy phenotype in motor neurons and patient cells. Strikingly, interaction of SETX with ZPR1 is disrupted in ALS4 patients that have heterozygous SETX (L389S) mutation. ZPR1 fails to recruit the mutant SETX homodimer but recruits the heterodimer with partially disrupted interaction between SETX and ZPR1. Interestingly, disruption of SETX-ZPR1 complexes causes increase in R-loop resolution activity leading to fewer R-loops in ALS4. Modulation of ZPR1 levels regulates R-loop accumulation and rescues the pathogenic R-loop phenotype in ALS4 patient cells. These findings originate a new concept, 'opposite alterations in a cell biological activity (R-loop resolution) result in similar pathogenesis (neurodegeneration) in different genetic motor neuron disorders'. We propose that ZPR1 collaborates with SETX and may function as a molecular brake to regulate SETX-dependent R-loop resolution activity critical for the normal functioning of motor neurons.

Essential Roles of the Smc5/6 Complex in Replication through Natural Pausing Sites and Endogenous DNA Damage Tolerance.

The essential functions of the conserved Smc5/6 complex remain elusive. To uncover its roles in genome maintenance, we established Saccharomyces cerevisiae cell-cycle-regulated alleles that enable restriction of Smc5/6 components to S or G2/M. Unexpectedly, the essential functions of Smc5/6 segregated fully and selectively to G2/M. Genetic screens that became possible with generated alleles identified processes that crucially rely on Smc5/6 specifically in G2/M: metabolism of DNA recombination structures triggered by endogenous replication stress, and replication through natural pausing sites located in late-replicating regions. In the first process, Smc5/6 modulates remodeling of recombination intermediates, cooperating with dissolution activities. In the second, Smc5/6 prevents chromosome fragility and toxic recombination instigated by prolonged pausing and the fork protection complex, Tof1-Csm3. Our results thus dissect Smc5/6 essential roles and reveal that combined defects in DNA damage tolerance and pausing site-replication cause recombination-mediated DNA lesions, which we propose to drive developmental and cancer-prone disorders.

Error-free DNA damage tolerance and sister chromatid proximity during DNA replication rely on the Polα/Primase/Ctf4 Complex.

Chromosomal replication is entwined with DNA damage tolerance (DDT) and chromatin structure establishment via elusive mechanisms. Here we examined how specific replication conditions affecting replisome architecture and repriming impact on DDT. We show that Saccharomyces cerevisiae Polα/Primase/Ctf4 mutants, proficient in bulk DNA replication, are defective in recombination-mediated damage-bypass by template switching (TS) and have reduced sister chromatid cohesion. The decrease in error-free DDT is accompanied by increased usage of mutagenic DDT, fork reversal, and higher rates of genome rearrangements mediated by faulty strand annealing. Notably, the DDT defects of Polα/Primase/Ctf4 mutants are not the consequence of increased sister chromatid distance, but are instead caused by altered single-stranded DNA metabolism and abnormal replication fork topology. We propose that error-free TS is driven by timely replicative helicase-coupled re-priming. Defects in this event impact on replication fork architecture and sister chromatid proximity, and represent a frequent source of chromosome lesions upon replication dysfunctions.

Rtt107 Is a Multi-functional Scaffold Supporting Replication Progression with Partner SUMO and Ubiquitin Ligases.

Elucidating the individual and collaborative functions of genome maintenance factors is critical for understanding how genome duplication is achieved. Here, we investigate a conserved scaffold in budding yeast, Rtt107, and its three partners: a SUMO E3 complex, a ubiquitin E3 complex, and Slx4. Biochemical and genetic findings show that Rtt107 interacts separately with these partners and contributes to their individual functions, including a role in replisome sumoylation. We also provide evidence that Rtt107 associates with replisome components, and both itself and its associated E3s are important for replicating regions far from initiation sites. Corroborating these results, replication defects due to Rtt107 loss and genotoxic sensitivities in mutants of Rtt107 and its associated E3s are rescued by increasing replication initiation events through mutating two master repressors of late origins, Mrc1 and Mec1. These findings suggest that Rtt107 functions as a multi-functional platform to support replication progression with its partner E3 enzymes.

Local regulation of the Srs2 helicase by the SUMO-like domain protein Esc2 promotes recombination at sites of stalled replication.

Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to replication forks, how recombination-mediated damage bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domain-containing Saccharomyces cerevisiae protein Esc2 facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recombinase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause suboptimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress and globally prevented at undamaged replicating chromosomes.

Selective modulation of the functions of a conserved DNA motor by a histone fold complex.

Budding yeast Mph1 helicase and its orthologs drive multiple DNA transactions. Elucidating the mechanisms that regulate these motor proteins is central to understanding genome maintenance processes. Here, we show that the conserved histone fold MHF complex promotes Mph1-mediated repair of damaged replication forks but does not influence the outcome of DNA double-strand break repair. Mechanistically, scMHF relieves the inhibition imposed by the structural maintenance of chromosome protein Smc5 on Mph1 activities relevant to replication-associated repair through binding to Mph1 but not DNA. Thus, scMHF is a function-specific enhancer of Mph1 that enables flexible response to different genome repair situations.

Error-free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin.

DNA damage tolerance (DDT) mechanisms facilitate replication resumption and completion when DNA replication is blocked by bulky DNA lesions. In budding yeast, template switching (TS) via the Rad18/Rad5 pathway is a favored DDT pathway that involves usage of the sister chromatid as a template to bypass DNA lesions in an error-free recombination-like process. Here, we establish that the Snf2 family translocase Irc5 is a novel factor that promotes TS and averts single-stranded DNA persistence during replication. We demonstrate that, during replication stress, Irc5 enables replication progression by assisting enrichment of cohesin complexes, recruited in an Scc2/Scc4-dependent fashion, near blocked replication forks. This allows efficient formation of sister chromatid junctions that are crucial for error-free DNA lesion bypass. Our results support the notion of a key role of cohesin in the completion of DNA synthesis under replication stress and reveal that the Rad18/Rad5-mediated DDT pathway is linked to cohesin enrichment at sites of perturbed replication via the Snf2 family translocase Irc5.

SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability.

SMC5/6 function in genome integrity remains elusive. Here, we show that SMC5 dysfunction in avian DT40 B cells causes mitotic delay and hypersensitivity toward DNA intra- and inter-strand crosslinkers (ICLs), with smc5 mutants being epistatic to FANCC and FANCM mutations affecting the Fanconi anemia (FA) pathway. Mutations in the checkpoint clamp loader RAD17 and the DNA helicase DDX11, acting in an FA-like pathway, do not aggravate the damage sensitivity caused by SMC5 dysfunction in DT40 cells. SMC5/6 knockdown in HeLa cells causes MMC sensitivity, increases nuclear bridges, micronuclei, and mitotic catastrophes in a manner similar and non-additive to FANCD2 knockdown. In both DT40 and HeLa systems, SMC5/6 deficiency does not affect FANCD2 ubiquitylation and, unlike FANCD2 depletion, RAD51 focus formation. SMC5/6 components further physically interact with FANCD2-I in human cells. Altogether, our data suggest that SMC5/6 functions jointly with the FA pathway to support genome integrity and DNA repair and may be implicated in FA or FA-related human disorders.

Maintaining genome stability at the replication fork.

Aberrant DNA replication is a major source of the mutations and chromosome rearrangements that are associated with pathological disorders. When replication is compromised, DNA becomes more prone to breakage. Secondary structures, highly transcribed DNA sequences and damaged DNA stall replication forks, which then require checkpoint factors and specialized enzymatic activities for their stabilization and subsequent advance. These mechanisms ensure that the local DNA damage response, which enables replication fork progression and DNA repair in S phase, is coupled with cell cycle transitions. The mechanisms that operate in eukaryotic cells to promote replication fork integrity and coordinate replication with other aspects of chromosome maintenance are becoming clear.

A cell cycle-regulated Slx4-Dpb11 complex promotes the resolution of DNA repair intermediates linked to stalled replication.

A key function of the cellular DNA damage response is to facilitate the bypass of replication fork-stalling DNA lesions. Template switch reactions allow such a bypass and involve the formation of DNA joint molecules (JMs) between sister chromatids. These JMs need to be resolved before cell division; however, the regulation of this process is only poorly understood. Here, we identify a regulatory mechanism in yeast that critically controls JM resolution by the Mus81-Mms4 endonuclease. Central to this regulation is a conserved complex comprising the scaffold proteins Dpb11 and Slx4 that is under stringent control. Cell cycle-dependent phosphorylation of Slx4 by Cdk1 promotes the Dpb11-Slx4 interaction, while in mitosis, phosphorylation of Mms4 by Polo-like kinase Cdc5 promotes the additional association of Mus81-Mms4 with the complex, thereby promoting JM resolution. Finally, the DNA damage checkpoint counteracts Mus81-Mms4 binding to the Dpb11-Slx4 complex. Thus, Dpb11-Slx4 integrates several cellular inputs and participates in the temporal program for activation of the JM-resolving nuclease Mus81.