Roles of the polymerase and BRCT domains of Rev1 protein in translesion DNA synthesis in yeast in vivo

Otsuka, Chie; Kunitomi, Nozomu; Iwai, Shigenori; Loakes, David; Negishi, Kazuo

doi:10.1016/j.mrfmmm.2005.03.005

Cited by 45 publications

(38 citation statements)

References 20 publications

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“…Insertion of cytosine residues opposite abasic sites by Rev1 constitutes over one-half of the bypass events in yeast (16)(17)(18). Therefore, we next investigated whether Rev1 was responsive to the modified clamp.…”

Section: Resultsmentioning

confidence: 99%

“…Rev1 is a deoxycytidyl transferase that shows the highest catalytic activity opposite template guanines and abasic sites (14,15). Rev1 is primarily responsible for inserting dC residues opposite abasic sites during mutagenesis (16)(17)(18). Several models have been proposed in which one DNA polymerase carries out the insertion step across the lesion, whereas a second polymerase extends from the lesion (19,20).…”

mentioning

confidence: 99%

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

Garg

Burgers

2005

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

225

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: 99%

mentioning

confidence: 99%

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

Garg

Burgers

2005

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

225

254

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“…Site-directed mutagenesis of D569 and E570 to alanine within the catalytic domain of this form of human REV1 inactivates the dCMP transferase activity [42]. When examined in yeast and chicken REV1, the dCMP transferase activity is not critical for damage tolerance activity [43,44]. Other structural domains are known to be important.…”

Section: Dna Pol ζ In Saccharomyces Cerevisiaementioning

confidence: 99%

DNA polymerase zeta (pol ζ) in higher eukaryotes

et al. 2007

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Most current knowledge about DNA polymerase zeta (pol ζ) comes from studies of the enzyme in the budding yeast Saccharomyces cerevisiae, where pol ζ consists of a complex of the catalytic subunit Rev3 with Rev7, which associates with Rev1. Most spontaneous and induced mutagenesis in yeast is dependent on these gene products, and yeast pol ζ can mediate translesion DNA synthesis past some adducts in DNA templates. Study of the homologous gene products in higher eukaryotes is in a relatively early stage, but additional functions for the eukaryotic proteins are already apparent. Suppression of vertebrate REV3L function not only reduces induced point mutagenesis but also causes larger-scale genome instability by raising the frequency of spontaneous chromosome translocations. Disruption of Rev3L function is tolerated in Drosophila, Arabidopsis, and in vertebrate cell lines under some conditions, but is incompatible with mouse embryonic development. Functions for REV3L and REV7(MAD2B) in higher eukaryotes have been suggested not only in translesion DNA synthesis but also in some forms of homologous recombination, repair of interstrand DNA crosslinks, somatic hypermutation of immunoglobulin genes and cell-cycle control. This review discusses recent developments in these areas.

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“…The yeast rev1 mutant displays a complete loss of mutagenesis activity comparable to that of rev3, which cannot be explained by its dCMP transferase activity. Indeed, analysis of site-specific mutations confirms that the Rev1 enzymatic activity is not essential for TLS, but its BRCA1 C-terminal (BRCT) domain [94,95] and/or a polymerase-associated domain (PAD) [96] are required for protein interactions. The C-terminal 100 amino acids of human Rev1 are sufficient to interact with all other TLS polymerases [97] ( Figure 3), implying a scaffold role of Rev1 in TLS.…”

Section: Y Family Dna Polymerasesmentioning

confidence: 99%

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

Andersen¹,

Xiao³

2007

Cell Res

185

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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Roles of the polymerase and BRCT domains of Rev1 protein in translesion DNA synthesis in yeast in vivo

Cited by 45 publications

References 20 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

DNA polymerase zeta (pol ζ) in higher eukaryotes

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

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