Mms1 and Mms22 stabilize the replisome during replication stress

Mms1 and Mms22 form a Cul4(Ddb1)-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101(Mms1/Mms22) ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1-Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.     

HUWE1 interacts with PCNA to alleviate replication stress

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S‐phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT‐type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA. We show that HUWE1‐knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono‐ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.     

SUMOylation mediates CtIP’s functions in DNA end resection and replication fork protection

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP’s function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP’s carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability.     

HUWE1 comes to the rescue at stalled replication forks

HUWE1 is a multi‐faceted E3 ubiquitin ligase of the HECT family with many confirmed substrates, but mechanistic understanding of its functional roles in signaling pathways remains limited. In this issue of EMBO Reports, Choe et al demonstrate a novel function for HUWE1 in promoting DNA damage tolerance mechanisms to bypass DNA lesions during replication stress, thereby preserving genome stability. The authors connect this role for HUWE1 with its function in maintaining H2AX monoubiquitination levels for efficient signaling at stalled replication forks 1 . Thus, this work highlights HUWE1 as a novel player in the replication stress response and prompts further investigation of its regulation during replication and other cellular processes.     

Spartan/C1orf124, a reader of PCNA ubiquitylation and a regulator of UV-induced DNA damage response

PCNA is a key component of DNA replication and repair machineries. DNA damage-induced PCNA ubiquitylation serves as a molecular mark to orchestrate postreplication repair. Here, we have identified and characterized Spartan, a protein that specifically recognizes ubiquitylated PCNA and plays an important role in cellular resistance to UV radiation. In vitro, Spartan engages ubiquitylated PCNA via both a PIP box and a UBZ domain. In cells, Spartan is recruited to sites of UV damage in a manner dependent upon the PIP box, the UBZ domain, and PCNA ubiquitylation. Furthermore, Spartan colocalizes and interacts with Rad18, the E3 ubiquitin ligase that modifies PCNA. Surprisingly, while Spartan is recruited by ubiquitylated PCNA, knockdown of Spartan compromised chromatin association of Rad18, monoubiquitylation of PCNA, and localization of Pol η to UV damage. Thus, as a "reader" of ubiquitylated PCNA, Spartan promotes an unexpected feed-forward loop to enhance PCNA ubiquitylation and translesion DNA synthesis.     

The F-box protein Dia2 overcomes replication impedance to promote genome stability in Saccharomyces cerevisiae

The maintenance of DNA replication fork stability under conditions of DNA damage and at natural replication pause sites is essential for genome stability. Here, we describe a novel role for the F-box protein Dia2 in promoting genome stability in the budding yeast Saccharomyces cerevisiae. Like most other F-box proteins, Dia2 forms a Skp1-Cdc53/Cullin-F-box (SCF) E3 ubiquitin–ligase complex. Systematic analysis of genetic interactions between dia2Δ and ~4400 viable gene deletion mutants revealed synthetic lethal/synthetic sick interactions with a broad spectrum of DNA replication, recombination, checkpoint, and chromatin-remodeling pathways. dia2Δ strains exhibit constitutive activation of the checkpoint kinase Rad53 and elevated counts of endogenous DNA repair foci and are unable to overcome MMS-induced replicative stress. Notably, dia2Δ strains display a high rate of gross chromosomal rearrangements (GCRs) that involve the rDNA locus and an increase in extrachromosomal rDNA circle (ERC) formation, consistent with an observed enrichment of Dia2 in the nucleolus. These results suggest that Dia2 is essential for stable passage of replication forks through regions of damaged DNA and natural fragile regions, particularly the replication fork barrier (RFB) of rDNA repeat loci. We propose that the SCF^Dia2 ubiquitin ligase serves to modify or degrade protein substrates that would otherwise impede the replication fork in problematic regions of the genome.     

RNF4 controls the extent of replication fork reversal to preserve genome stability

Replication fork reversal occurs via a two-step process that entails reversal initiation and reversal extension. DNA topoisomerase IIalpha (TOP2A) facilitates extensive fork reversal, on one hand through resolving the topological stress generated by the initial reversal, on the other hand via its role in recruiting the SUMO-targeted DNA translocase PICH to stalled forks in a manner that is dependent on its SUMOylation by the SUMO E3 ligase ZATT. However, how TOP2A activities at stalled forks are precisely regulated remains poorly understood. Here we show that, upon replication stress, the SUMO-targeted ubiquitin E3 ligase RNF4 accumulates at stalled forks and targets SUMOylated TOP2A for ubiquitination and degradation. Downregulation of RNF4 resulted in aberrant activation of the ZATT–TOP2A–PICH complex at stalled forks, which in turn led to excessive reversal and elevated frequencies of fork collapse. These results uncover a previously unidentified regulatory mechanism that regulates TOP2A activities at stalled forks and thus the extent of fork reversal.     

Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63-linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)-induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.     

Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates

Oncogene-induced DNA replication stress activates the DNA damage response (DDR), a crucial anticancer barrier. DDR inactivation in these conditions promotes genome instability and tumor progression, but the underlying molecular mechanisms are elusive. We found that overexpression of both Cyclin E and Cdc25A rapidly slowed down replication forks and induced fork reversal, suggestive of increased topological stress. Surprisingly, these phenotypes, per se, are neither associated with chromosomal breakage nor with significant DDR activation. Oncogene-induced DNA breakage and DDR activation instead occurred upon persistent G2/M arrest or, in a checkpoint-defective context, upon premature CDK1 activation. Depletion of MUS81, a cell cycle–regulated nuclease, markedly limited chromosomal breakage and led to further accumulation of reversed forks. We propose that nucleolytic processing of unusual replication intermediates mediates oncogene-induced genotoxicity and that limiting such processing to mitosis is a central anti-tumorigenic function of the DNA damage checkpoints.     

WRNIP1 protects stalled forks from degradation and promotes fork restart after replication stress

Accurate handling of stalled replication forks is crucial for the maintenance of genome stability. RAD51 defends stalled replication forks from nucleolytic attack, which otherwise can threaten genome stability. However, the identity of other factors that can collaborate with RAD51 in this task is poorly elucidated. Here, we establish that human Werner helicase interacting protein 1 (WRNIP1) is localized to stalled replication forks and cooperates with RAD51 to safeguard fork integrity. We show that WRNIP1 is directly involved in preventing uncontrolled MRE11‐mediated degradation of stalled replication forks by promoting RAD51 stabilization on ssDNA. We further demonstrate that replication fork protection does not require the ATPase activity of WRNIP1 that is however essential to achieve the recovery of perturbed replication forks. Loss of WRNIP1 or its catalytic activity causes extensive DNA damage and chromosomal aberrations. Intriguingly, downregulation of the anti‐recombinase FBH1 can compensate for loss of WRNIP1 activity, since it attenuates replication fork degradation and chromosomal aberrations in WRNIP1‐deficient cells. Therefore, these findings unveil a unique role for WRNIP1 as a replication fork‐protective factor in maintaining genome stability.     

Chromosome demise in the wake of ligase-deficient replication

Bacterial DNA ligases, NAD⁺‐dependent enzymes, are distinct from eukaryotic ATP‐dependent ligases, representing promising targets for broad‐spectrum antimicrobials. Yet, the chromosomal consequences of ligase‐deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase‐deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double‐strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non‐allelic double‐strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double‐strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase‐deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double‐strand breaks and then into irreparable double‐strand gaps may be behind lethality of any DNA damaging treatment.     

RAD18 lives a double life: Its implication in DNA double-strand break repair

Maintenance of genome stability depends on efficient and accurate repair of DNA lesions. Failure to properly repair damaged DNA can cause cell death, mutations and chromosomal instability, which eventually lead to tumorigenesis. The E3 ligase RAD18 is well-known for its function in DNA damage bypass and post-replication repair (PRR) in yeast and vertebrates via its ability to facilitate PCNA mono-ubiquitination at stalled replication forks. However, emerging evidence has also indicated that RAD18 plays an important role in homologous recombination (HR) in mammalian cells, which is an error-free DNA repair pathway that mediates the repair of double-strand breaks (DSBs). Here, we review how RAD18 carries out these distinct functions in response to different types of DNA lesions.     

Critical roles of ring finger protein RNF8 in replication stress responses

Histone ubiquitylation is emerging as an important protective component in cellular responses to DNA damage. The ubiquitin ligases RNF8 and RNF168 assemble ubiquitin chains onto histone molecules surrounding DNA breaks and facilitate retention of DNA repair proteins. Although RNF8 and RNF168 play important roles in repair of DNA double strand breaks, their requirement for cell protection from replication stress is largely unknown. In this study, we uncovered RNF168-independent roles of RNF8 in repair of replication inhibition-induced DNA damage. We showed that RNF8 depletion, but not RNF168 depletion, hyper-sensitized cells to hydroxyurea and aphidicolin treatment. Consistently, hydroxyurea induced persistent single strand DNA lesions and sustained CHK1 activation in RNF8-depleted cells. In line with strict requirement for RAD51-dependent repair of hydroxyurea-stalled replication forks, RNF8 depletion compromised RAD51 accumulation onto single strand DNA lesions, suggesting that impaired replication fork repair may underlie the enhanced cellular sensitivity to replication arrest observed in RNF8-depleted cells. In total, our study highlights the differential requirement for the ubiquitin ligase RNF8 in facilitating repair of replication stress-associated DNA damage.     

Regulation of gross chromosomal rearrangements by ubiquitin and SUMO ligases in Saccharomyces cerevisiae

Gross chromosomal rearrangements (GCRs) are frequently observed in many cancers. Previously, we showed that inactivation of Rad5 or Rad18, ubiquitin ligases (E3) targeting for proliferating cell nuclear antigen (PCNA), increases the de novo telomere addition type of GCR (S. Smith, J. Y. Hwang, S. Banerjee, A. Majeed, A. Gupta, and K. Myung, Proc. Natl. Acad. Sci. USA 101:9039-9044, 2004). GCR suppression by Rad5 and Rad18 appears to be exerted by the RAD5-dependent error-free mode of bypass DNA repair. In contrast, Siz1 SUMO ligase and another ubiquitin ligase, Bre1, which target for PCNA and histone H2B, respectively, have GCR-supporting activities. Inactivation of homologous recombination (HR) proteins or the helicase Srs2 reduces GCR rates elevated by the rad5 or rad18 mutation. GCRs are therefore likely to be produced through the restrained recruitment of an HR pathway to stalled DNA replication forks. Since this HR pathway is compatible with Srs2, it is not a conventional form of recombinational pathway. Lastly, we demonstrate that selection of proper DNA repair pathways to stalled DNA replication forks is controlled by the Mec1-dependent checkpoint and is executed by cooperative functions of Siz1 and Srs2. We propose a mechanism for how defects in these proteins could lead to diverse outcomes (proper repair or GCR formation) through different regulation of DNA repair machinery.     

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure.     

Recql5 plays an important role in DNA replication and cell survival after camptothecin treatment

Disruption of replication can lead to loss of genome integrity and increase of cancer susceptibility in mammals. Thus, a replication impediment constitutes a formidable challenge to these organisms. Recent studies indicate that homologous recombination (HR) plays an important role in suppressing genome instability and promoting cell survival after exposure to various replication inhibitors, including a topoisomerase I inhibitor, camptothecin (CPT). Here, we report that the deletion of RecQ helicase Recql5 in mouse ES cells and embryonic fibroblast (MEF) cells resulted in a significant increase in CPT sensitivity and a profound reduction in DNA replication after the treatment with CPT, but not other DNA-damaging agents. This CPT-induced cell death is replication dependent and occurs primarily after the cells had exited the first cell cycle after CPT treatment. Furthermore, we show that Recql5 functions nonredundantly with Rad51, a key factor for HR to protect mouse ES cells from CPT-induced cytotoxicity. These new findings strongly suggest that Recql5 plays an important role in maintaining active DNA replication to prevent the collapse of replication forks and the accumulation of DSBs in order to preserve genome integrity and to prevent cell death after replication stress as a result of topoisomerase I poisoning.     

Direct role for proliferating cell nuclear antigen in substrate recognition by the E3 ubiquitin ligase CRL4Cdt2

The E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) is emerging as an important cell cycle regulator that targets numerous proteins for destruction in S phase and after DNA damage, including Cdt1, p21, and Set8. CRL4(Cdt2) substrates contain a "PIP degron," which consists of a canonical proliferating cell nuclear antigen (PCNA) interaction motif (PIP box) and an adjacent basic amino acid. Substrates use their PIP box to form a binary complex with PCNA on chromatin and the basic residue to recruit CRL4(Cdt2) for substrate ubiquitylation. Using Xenopus egg extracts, we identify an acidic residue in PCNA that is essential to support destruction of all CRL4(Cdt2) substrates. This PCNA residue, which adjoins the basic amino acid of the bound PIP degron, is dispensable for substrate binding to PCNA but essential for CRL4(Cdt2) recruitment to chromatin. Our data show that the interaction of CRL4(Cdt2) with substrates requires molecular determinants not only in the substrate degron but also on PCNA. The results illustrate a potentially general mechanism by which E3 ligases can couple ubiquitylation to the formation of protein-protein interactions.     

Integrating DNA replication with trans-lesion synthesis via Cdc7

It has long been appreciated that Cdc7 is an essential protein kinase that phosphorylates Mcm2-7 helicase subunits to promote initiation of DNA replication. In addition to its well-elucidated role in DNA replication, recent studies suggest that DDK is active in genotoxin-treated cells and may mediate aspects of the DNA damage response. However, specific role(s) of DDK and its effector targets in DNA damage signaling have not been defined. A recent study from our laboratories has identified the E3 ubiquitin ligase Rad18 as novel substrate of DDK in vitro and in human cells. Rad18 plays a central role in a post-replication DNA repair pathway termed ‘Trans-Lesion Synthesis’ (TLS) by promoting recruitment of DNA Polymerase eta (Polη) and other TLS polymerases to stalled replication forks. DDK-mediated Rad18 phosphorylation promotes Rad18-Polη complex formation and facilitates Rad18-dependent recruitment of Polη to stalled replication forks. The mechanisms that regulate Rad18-dependent TLS are incompletely understood. Our study provides the first demonstration of Rad18 regulation by direct phosphorylation and defines a novel mechanism for Rad18-dependent recruitment of TLS polymerases to stalled forks. This study also demonstrates a molecular basis for integration of TLS with S-phase progression via the essential Cdc7 kinase. These findings reveal unexpected mechanistic insights to the regulation of the TLS pathway and Polη recruitment.     

Interplay between Np95 and Eme1 in the DNA damage response

Mus81 (methyl methansulfonate UV sensitive clone 81) and Eme1 (essential meiotic endonuclease 1, also known as MMS4) form a heterodimeric endonuclease that is critical for genomic stability and the response to DNA crosslink damage and replication blockade. However, relatively little is known as to how this endonuclease is regulated following DNA damage. Here, we report mammalian Eme1 interacts with Np95, an E3 ubiquitin ligase that participates in chromatin modification, replication-linked epigenetic maintenance and the DNA damage response. Np95 and Eme1 co-localize on nuclear chromatin following exposure of cells to camptothecin, an agent that promotes the collapse of replication forks. The observed co localization following DNA damage was found to be dependent on an intact RING finger, the structural motif that encodes the E3 ubiquitin ligase activity of Np95. Taken together, these findings link Mus81-Eme1 with the replication-associated chromatin modifier functions of Np95 in the cellular response to DNA damage.