Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.

Torres‐Ramos, Carlos A.; Yoder, Bonita L.; Burgers, Peter M.; Prakash, Satya; Prakash, Louise

doi:10.1073/pnas.93.18.9676

Cited by 96 publications

(71 citation statements)

References 42 publications

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“…The damage avoidance pathway of postreplication DNA repair is activated by Lys-63-linked polyubiquitination of monoubiquitinated PCNA in a Rad5͞Mms2͞Ubc13-dependent manner (1). We have previously identified a PCNA mutant (pol30-46) that is still functional for TLS but is defective for the damage avoidance branch (35). As expected, this mutant PCNA was still monoubiquitinated like wild type (Fig.…”

Section: Resultsmentioning

confidence: 61%

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1

Garg

Burgers

2005

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

223

254

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In response to DNA damage, the Rad6͞Rad18 ubiquitin-conjugating complex monoubiquitinates the replication clamp proliferating cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast PCNA specifically on Lys-164. Significantly, only those PCNA clamps that are appropriately loaded around effector DNA by its loader, replication factor C, are ubiquitinated. This observation suggests that, in vitro, only PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated PCNA displays the same replicative functions as unmodified PCNA. These functions include loading onto DNA by replication factor C, as well as Okazaki fragment synthesis and maturation by the PCNA-coordinated actions of DNA polymerase ␦, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase , the yeast Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.DNA replication ͉ postreplication repair ͉ translesion synthesis ͉ ubiquitination ͉ yeast M odification of the replication clamp proliferating cell nuclear antigen (PCNA) has recently emerged as an important regulatory switch during DNA replication and DNA damage response. The critical site of modification on PCNA is Lys-164, and either sumoylation or ubiquitination at this residue can occur (1, 2). Although sumoylation at Lys-164 is proposed to be associated with normal replicative functions, monoubiquitination of this residue by the Rad6͞Rad18 ubiquitin E2͞E3 complex channels DNA damage into the postreplication DNA repair (PRR) pathway (1,3,4). The PRR pathway is comprised of two error-prone branches involving translesion synthesis (TLS) by error-prone DNA polymerases, and an error-free damage avoidance branch that proceeds by fork regression and template switching (5, 6). The latter branch depends on further modification of monoubiquitinated PCNA involving Rad5 and Mms2͞Ubc13 E2͞E3 catalyzed formation of polyubiquitin chains via an unusual Lys-63 ubiquitin linkage (1).Replicative DNA polymerases of the B-family possess a very restrictive active site, which promotes high-fidelity DNA replication but inhibits bypass synthesis of many DNA lesions (5,7,8). On the other hand, Y-family DNA polymerases are particularly adapted to the bypass of DNA lesions because of a more open active site. For instance, DNA polymerase (Pol ), the ortholog of the human Xeroderma pigmentosum protein, can accommodate a cis-syn pyrimidine dimer in its active site allowing facile damage bypass (9). TLS in yeast ...

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Section: Resultsmentioning

confidence: 61%

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1

Garg

Burgers

2005

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

223

254

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“…In addition to acting as a permissivity factor for replication-related DNA synthesis, PCNA plays a role in synthesis related to DNA damage repair (Torres-Ramos et al, 1996). Associated with translocation of BRCA1 to these PCNA-containing complexes, BRCA1 was phosphorylated, to a greater extent than normally occurs during entry into S-phase (Scully et al, 1997c).…”

Section: Functional Activities Of Brca1 Cell Cycle Regulation and Gromentioning

confidence: 99%

BRCA1 gene in breast cancer

Rosen

Fan

Pestell

et al. 2003

Journal Cellular Physiology

237

195

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The BRCA1 gene was identified and cloned in 1994 based on its linkage to early onset breast cancer and breast-ovarian cancer syndromes in women. While inherited mutations of BRCA1 are responsible for about 40-45% of hereditary breast cancers, these mutations account for only 2-3% of all breast cancers, since the BRCA1 gene is rarely mutated in sporadic breast cancers. However, BRCA1 expression is frequently reduced or absent in sporadic cancers, suggesting a much wider role in mammary carcinogenesis. Since BRCA1 was cloned in 1994, its molecular function has been the subject of intense investigation. These studies have revealed multiple functions of the BRCA1 that may contribute to its tumor suppressor activity, including roles in: cell cycle progression, several highly specialized DNA repair processes, DNA damage-responsive cell cycle checkpoints, regulation of a set of specific transcriptional pathways, and apoptosis. Many of these functions are linked to protein:protein interactions involving different portions of the 1,863 amino acid (aa) BRCA1 protein. BRCA1 functions in cell cycle progression and the DNA damage response appear to be regulated by distinct and specific phosphorylation events, but the molecular pathways activated by these phosphorylations are only beginning to be unraveled. In addition, the reason that BRCA1 mutation carriers develop specific tumor types (breast and ovarian cancers in women and possibly prostate cancers in men) is not clearly understood. Elucidation of the precise molecular functions of the BRCA1 gene product will greatly enhance our understanding of the pathogenesis of hereditary as well as sporadic mammary carcinogenesis.

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“…pol30-46 is epistatic to rad6 and rad18, but synergistic with rev3. The pol30-46 mutant is normal in UV-induced mutagenesis and DNA synthesis but displays significantly reduced PRR activity as judged by the alkaline sedimentation assay [61].…”

Section: Ddt In Saccharomyces Cerevisiaementioning

confidence: 99%

“…The involvement of this DNA-polymerase sliding clamp in DDT was first suggested by the isolation and characterization of the pol30-46 allele [61]. pol30-46 is epistatic to rad6 and rad18, but synergistic with rev3.…”

Section: Ddt In Saccharomyces Cerevisiaementioning

confidence: 99%

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

Andersen¹,

Xiao³

2007

Cell Res

184

187

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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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Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.

Cited by 96 publications

References 42 publications

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1

BRCA1 gene in breast cancer

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

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