Rad18 Regulates DNA Polymerase κ and Is Required for Recovery from S-Phase Checkpoint-Mediated Arrest

Bi, Xin; Barkley, Laura R.; Slater, Damien; Tateishi, Seiichiro; Yamaizumi, Masaru; Ohmori, Hitoshi; Vaziri, Cyrus

doi:10.1128/mcb.26.9.3527-3540.2006

Cited by 154 publications

(201 citation statements)

References 47 publications

(72 reference statements)

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“…Unlike other Y-family polymerases, Rev1 does not contain a PIP motif; instead, a recent study npg suggests that Rev1 utilizes its BRCT domain to interact with PCNA [95]. Supporting this notion is the observation that the damage-induced foci formation of Polη [122,124] and Polκ [125] is dependent on functional Rad18, presumably because Rad18 is required for the generation of monoubiquitinated PCNA. An ultimate support perhaps comes from an in vitro study [126], in which PCNA was found to be only ubiquitinated when appropriately loaded onto DNA.…”

Section: Regulated Access Of Y-family Polymerases To the Damage Sitementioning

confidence: 52%

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Andersen¹,

Xiao³

2007

Cell Res

183

186

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npg In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance (DDT) has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen (PCNA) by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis (TLS) with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer. DNA damage toleranceIn the presence of spontaneous or carcinogen-induced DNA damage, living cells have to maintain and complete DNA synthesis or risk replication fork collapse. Since the process of DNA licensing is to ensure the genome is duplicated once and only once during each cell cycle, stalled or collapsed replication forks may not be able to restart, which often results in double-strand breaks (DSBs) and causes compromised genome integrity or cell death. In addition to highly conserved DNA repair pathways, all living organisms have evolved schemes to ensure continuation of DNA synthesis in the presence of damage. These schemes were originally termed DNA postreplication repair (PRR) due to observations of transient shortened nascent DNA structures following S phase in response to DNA damage. In bacteria and unicellular yeast, these shortened DNA segments can be measured by an alkaline sedimentation assay [1] or directly observed in electron micrographs [2]. In wild-type cells, these truncated DNA segments were restored to full length following a short recovery time. One typical experiment [1] involved the restoration of the nascent strand following UV exposure in nucleotide excision repair (NER)-deficient cells and was originally assumed to represent a mechanism of DNA repair. However, further investigation revealed that, although the nascent fragments were re-annealed, the original UV-induced pyrimidine dimers, which were responsible for the generation of single-strand gaps, often persisted in the genome [3,4]. It was argued that the replication-blocking lesion was not necessarily corrected, but rather transiently bypassed and carried over to the next generation. Perhaps it is more beneficial for the organi...

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Section: Regulated Access Of Y-family Polymerases To the Damage Sitementioning

confidence: 52%

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Andersen¹,

Xiao³

2007

Cell Res

183

186

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“…Also, a link between PCNA ubiquitination and checkpoint proteins was documented when using other DNA damaging agents. Vaziri and colleagues reported the requirement of RPA, ATR, and Chk1 for PCNA ubiquitination after treatment of cells with the bulky adduct-forming genotoxin benzo(a)pyrene dihydrodiol epoxide (20). More intriguingly, not only are ATR and Chk1 required for efficient activation of TLSassociated events, but also the TLS pol η promote efficient Chk1 activation.…”

Section: Discussionmentioning

confidence: 99%

“…The recruitment of specialized pols to replication factories can be monitored by analyzing their subnuclear organization by using high-resolution microscopy. This parameter is used as an indicator of TLS activation, given that the percentage of cells with detectable pol η nuclear foci steeply increases after UV irradiation (19)(20)(21)(22). To address the impact of ATR and Chk1 knockdown on pol η recruitment, we scored the nuclear pattern of pol η in two arbitrary categories: negative (none to 15 foci per cell) and positive (>15 foci per cell) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Kinase-independent function of checkpoint kinase 1 (Chk1) in the replication of damaged DNA

Speroni

Federico

Mansilla

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The checkpoint kinases Chk1 and ATR are broadly known for their role in the response to the accumulation of damaged DNA. Because Chk1 activation requires its phosphorylation by ATR, it is expected that ATR or Chk1 down-regulation should cause similar alterations in the signals triggered by DNA lesions. Intriguingly, we found that Chk1, but not ATR, promotes the progression of replication forks after UV irradiation. Strikingly, this role of Chk1 is independent of its kinase-domain and of its partnership with Claspin. Instead, we demonstrate that the ability of Chk1 to promote replication fork progression on damaged DNA templates relies on its recently identified proliferating cell nuclear antigen-interacting motif, which is required for its release from chromatin after DNA damage. Also supporting the importance of Chk1 release, a histone H2B-Chk1 chimera, which is permanently immobilized in chromatin, is unable to promote the replication of damaged DNA. Moreover, inefficient chromatin dissociation of Chk1 impairs the efficient recruitment of the specialized DNA polymerase η (pol η) to replication-associated foci after UV. Given the critical role of pol η during translesion DNA synthesis (TLS), these findings unveil an unforeseen facet of the regulation by Chk1 of DNA replication. This kinase-independent role of Chk1 is exclusively associated to the maintenance of active replication forks after UV irradiation in a manner in which Chk1 release prompts TLS to avoid replication stalling. T he checkpoint kinases ATR and Chk1 are central factors in the DNA damage response (1). During the S phase checkpoint, ATR is activated at single-stranded DNA (ssDNA) and this event, in turn, activates the effector kinase Chk1. Although ATR remains associated with the DNA, activated Chk1 rapidly spreads throughout the whole nucleus. Within the nucleoplasm, Chk1 delays the progression through S phase via phosphorylation of key target genes (2, 3).Several lines of evidence suggest that the activities of ATR and Chk1 are also required for proper unperturbed S phase progression. In fact, ATR or Chk1 loss leads to embryonic lethality (4-7), and Chk1 heterozygosity is associated with multiple defects, including a miscoordinated cell cycle and increased apoptosis (8).A contribution of Chk1 to replication fork stability during unperturbed DNA replication was identified and characterized in detail (9-11). Chk1 activity promotes the maintenance of global replication rates by regulating origin firing. In line with these observations, the monoallelic expression of the mutant Chk1 S317A, which is not phosphorylated by ATR, impairs fork elongation (12). Together, these results reveal an unambiguous role of the Chk1 kinase during unperturbed DNA replication. Intriguingly, recent reports described a kinase-independent effect of Chk1 on DNA replication-associated events. Scorah and colleagues discovered a proliferating cell nuclear antigen (PCNA) binding motif of Chk1 (Chk1_TRFF motif) required for the efficient dissociation of Chk1 from chromatin a...

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“…2, B-C) nor does loss of PCNA prevent ATR-dependent phosphorylation of Chk1 (24) (data not shown). Although PCNA ubiquitination is not dependent on the homolog of ATR, Rad3, in Schizosaccharomyces pombe (24), results in mammalian cells suggest a partial dependence on ATR, Chk1, and RPA (40). These findings could reflect a difference between yeast, Xenopus, and mammalian cells, although it is also conceivable that in mammalian cells the loss of ATR, which leads to genomic instability and cell death (41)(42)(43), perturbs the cell cycle and DNA replication over the course of this experiment.…”

Section: Discussionmentioning

confidence: 99%

Monoubiquitination of Proliferating Cell Nuclear Antigen Induced by Stalled Replication Requires Uncoupling of DNA Polymerase and Mini-chromosome Maintenance Helicase Activities

Chang

Lupardus

Cimprich

2006

Journal of Biological Chemistry

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Proliferating cell nuclear antigen (PCNA) is a homotrimeric, ring-shaped protein complex that functions as a processivity factor for DNA polymerases. Following genotoxic stress, PCNA is modified at a conserved site by either a single ubiquitin moiety or a polyubiquitin chain. These modifications are required to coordinate DNA damage tolerance processes with ongoing replication. The molecular mechanisms responsible for inducing PCNA ubiquitination are not well understood. Using Xenopus egg extracts, we show that ultraviolet radiation and aphidicolin treatment induce the mono-and diubiquitination of PCNA. PCNA ubiquitination is replication-dependent and coincides with activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent DNA damage checkpoint pathway. However, loss of ATR signaling by depletion of the ATR-interacting protein (ATRIP) or Rad1, a component of the 911 checkpoint clamp, does not impair PCNA ubiquitination. Primed single-stranded DNA generated by uncoupling of mini-chromosome maintenance helicase and DNA polymerase activities has been shown previously to be necessary for ATR activation. Here we show that PCNA ubiquitination also requires uncoupling of helicase and polymerase activities. We further demonstrate that replicating single-stranded DNA, which mimics the structure produced upon uncoupling, is sufficient to induce PCNA monoubiquitination. Our results suggest that PCNA ubiquitination and ATR activation are two independent events that occur in response to a common single-stranded DNA intermediate generated by functional uncoupling of mini-chromosome maintenance (MCM) helicase and DNA polymerase activities.Bulky lesions in the DNA cause arrest of replicative polymerases, halting cell cycle progression and activating DNA damage checkpoints. DNA damage tolerance mechanisms allow the cell to continue replication under these conditions and permit repair of the damage at a later time (1). Translesion synthesis (TLS) 2 is one type of DNA damage tolerance mechanism that allows specialized, low fidelity DNA polymerases to bypass DNA damage in an error-prone fashion. A second mechanism of DNA damage tolerance involves error-free strategies of bypass that include post-replication recombinational repair and replication fork regression. How these DNA damage tolerance mechanisms are coordinated with replication at stalled forks is largely unknown.One protein that plays a critical role in linking these processes is proliferating cell nuclear antigen (PCNA). PCNA is a homotrimeric ring-shaped protein complex that functions as a processivity factor necessary to maintain ongoing synthesis of DNA. PCNA has critical functions in DNA replication and DNA damage tolerance, and these functions depend on its interaction with other proteins. In addition, post-translational modification of PCNA has emerged as a critical step in regulating PCNA functions (1, 2).In response to DNA damage or replication fork stalling, PCNA is modified at a conserved site, Lys-164, by either a single ubiquitin moiet...

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Rad18 Regulates DNA Polymerase κ and Is Required for Recovery from S-Phase Checkpoint-Mediated Arrest

Cited by 154 publications

References 47 publications

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

Kinase-independent function of checkpoint kinase 1 (Chk1) in the replication of damaged DNA

Monoubiquitination of Proliferating Cell Nuclear Antigen Induced by Stalled Replication Requires Uncoupling of DNA Polymerase and Mini-chromosome Maintenance Helicase Activities

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