Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene

Clark, Donald R.

doi:10.1093/nar/gkg725

Cited by 38 publications

(37 citation statements)

References 28 publications

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“…However, no significant difference was observed between REV1-depleted cells and control cells (data not shown), indicating that it is difficult to assess effects of Hsp90 inhibition on REV1 function using this assay. These results are consistent with previous observations, which showed that antisense or ribozyme-mediated suppression of REV1 levels significantly decreased UVinduced mutagenesis but had little effects on UV-induced cytotoxicity (8,12). Thus, the mutagenesis assay seems more sensitive to reduced REV1 protein levels compared with the cell survival assay.…”

Section: Resultssupporting

confidence: 93%

Molecular Chaperone Hsp90 Regulates REV1-Mediated Mutagenesis

Pozo

Oda

Sekimoto

et al. 2011

Molecular and Cellular Biology

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REV1 is a Y-family polymerase that plays a central role in mutagenic translesion DNA synthesis (TLS), contributing to tumor initiation and progression. In a current model, a monoubiquitinated form of the replication accessory protein, proliferating cell nuclear antigen (PCNA), serves as a platform to recruit REV1 to damaged sites on the DNA template. Emerging evidence indicates that posttranslational mechanisms regulate REV1 in yeast; however, the regulation of REV1 in higher eukaryotes is poorly understood. Here we show that the molecular chaperone Hsp90 is a critical regulator of REV1 in human cells. Hsp90 specifically binds REV1 in vivo and in vitro. Treatment with a specific inhibitor of Hsp90 reduces REV1 protein levels in several cell types through proteasomal degradation. This is associated with suppression of UV-induced mutagenesis. Furthermore, Hsp90 inhibition disrupts the interaction between REV1 and monoubiquitinated PCNA and suppresses UV-induced focus formation. These results indicate that Hsp90 promotes folding of REV1 into a stable and/or functional form(s) to bind to monoubiquitinated PCNA. The present findings reveal a novel role of Hsp90 in the regulation of TLS-mediated mutagenesis.Genomic DNA is constantly exposed to both extrinsic and intrinsic genotoxic agents, such as UV light and oxidative stress. Although most DNA lesions are removed by multiple DNA repair pathways, some escape these mechanisms and persist in the genome. Such unrepaired DNA lesions usually block the progression of replication forks catalyzed by highfidelity DNA polymerases (Pols), which may lead to downstream reinitiation of DNA synthesis, leaving single-stranded DNA gaps. Translesion synthesis (TLS) is an essential mechanism for bypassing such replication blocks and postreplicative gaps by employing specialized Pols, including Pol , Pol , Pol , and REV1, members of the Y-family Pols (Y-Pols), and Pol , a member of the B family (10,14,28,43,58). These TLS Pols display low stringency for the active site and a lack of proofreading, and thus they contribute to mutagenesis and to DNA damage tolerance. Recruitment of the TLS Pols at sites of DNA damage depends on Rad6/Rad18-and CRL4Cdt2 -mediated production of monoubiquitinated proliferating cell nuclear antigen (PCNA) (Ub-PCNA), to which Y-Pols bind with high affinity (3, 21, 54, 57).REV1 plays a central role in promoting mutagenesis in lower and higher eukaryotes. Of note, the mutagenic activity of REV1 contributes to tumor initiation and progression in mammals (9, 29, 62). REV1 has a unique deoxycytidyl transferase activity in vitro, incorporating dCMP opposite several types of DNA lesions, such as abasic sites and damaged guanines (17,18,30,38,65); however, this enzymatic activity is dispensable for REV1-mediated TLS (37). Instead, the function of REV1 depends on protein-protein interactions via three key domains: the N-terminal BRCT domain (named after the C-terminal domain of a breast cancer susceptibility protein) (15,22,23), ubiquitin (Ub)-binding motifs (UBMs) in th...

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Section: Resultssupporting

confidence: 93%

Molecular Chaperone Hsp90 Regulates REV1-Mediated Mutagenesis

Pozo

Oda

Sekimoto

et al. 2011

Molecular and Cellular Biology

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“…In DT40 cells REV1 not only facilitates Polf-dependent bypass, but also modifies the catalytic behavior of Polf, restraining its synthetic activity to ensure that it incorporates nucleotides in-frame with the damaged template [125]. Human cell lines expressing high levels of human REV1 antisense RNA or ribozyme exhibit a much reduced frequency of 6-thioguanine-resistant mutants induced by UV-irradiation or BPDE [122,126,127], indicating that REV1 in higher eukaryotic cells performs functions similar to its yeast homologs. This property has been confirmed in a different experimental system using RNA interference to down-regulate mouse REV1 [128].…”

Section: Rev1 Functions In Vivomentioning

confidence: 99%

Y-family DNA polymerases in mammalian cells

Guo

Kosarek-Stancel

Tang

et al. 2009

Cell. Mol. Life Sci.

134

159

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Eukaryotic genomes are replicated with high fidelity to assure the faithful transmission of genetic information from one generation to the next. The accuracy of replication relies heavily on the ability of replicative DNA polymerases to efficiently select correct nucleotides for the polymerization reaction and, using their intrinsic exonuclease activities, to excise mistakenly incorporated nucleotides. Cells also possess a variety of specialized DNA polymerases that, by a process called translesion DNA synthesis (TLS), help overcome replication blocks when unrepaired DNA lesions stall the replication machinery. This review considers the properties of the Y-family (a subset of specialized DNA polymerases) and their roles in modulating spontaneous and genotoxic-induced mutations in mammals. We also review recent insights into the molecular mechanisms that regulate PCNA monoubiquitination and DNA polymerase switching during TLS and discuss the potential of using Y-family DNA polymerases as novel targets for cancer prevention and therapy.

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“…We have reported that many of the mutations induced by DDP adducts seem to result from error-prone translesional DNA synthesis mediated by DNA polymerase and/or hREV1 (Wu et al, 2004;Okuda et al, 2005). Several investigators have reported previously that a loss of hREV1 function markedly reduces UV-induced hypoxanthine guanine phosphoribosyl transferase mutations in human cells engineered to contain reduced levels of hREV1 mRNA through the expression of an hREV1 antisense RNA (Gibbs et al, 2000) or a ribozyme that cleaves endogenous hREV1 mRNA (Clark et al, 2003). It has also been demonstrated that inactivation of the REV1 gene in chicken DT40 cells renders them hypersensitive to a wide variety of mutagens, including DDP (Simpson and Sale, 2003).…”

mentioning

confidence: 99%

Human REV1 Modulates the Cytotoxicity and Mutagenicity of Cisplatin in Human Ovarian Carcinoma Cells

Lin

Okuda²,

Trang³

et al. 2006

Mol Pharmacol

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REV1 interacts with Y-type DNA polymerases (Pol) and Pol to bypass many types of adducts that block the replicative DNA polymerases. This pathway accounts for many of the mutations induced by cisplatin (cis-diamminedichloroplatinium II, DDP). This study sought to determine how increasing human REV1 (hREV1) affects the cytotoxicity and mutagenicity of DDP. Human ovarian carcinoma 2008 cells were transfected with an hREV1 expression vector and 4 sublines developed in which the hREV1 mRNA level was increased by 6.3-to 23.4-fold and hREV1 protein by 2.7-to 6.2-fold. The sublines were 1.3-to 1.7-fold resistant to the cytotoxic effect of DDP and 2.3-to 5.1-fold hypersensitive to the mutagenic effect of DDP. The hREV1-transfected sublines were 1.5-to 1.8-fold better than the parental 2008 cells at managing DDP adducts as assessed by their ability to express Renilla reniformis luciferase from a vector that had been extensively loaded with DDP adducts before transfection. Increased hREV1 expression was associated with a 1.5-fold increase in the rate at which the whole population acquired resistance to DDP during sequential cycles of drug exposure. Increasing the abundance of hREV1 thus resulted in both resistance to DDP and a significant elevation in DDP-induced mutagenicity. This was accompanied by an enhanced capacity to synthesize a functional protein from a DDPdamaged gene and, most importantly, by more rapid development of resistance during sequential cycles of DDP exposure that mimic clinical schedules of DDP administration. We conclude that hREV1-dependent processes are important determinants of DDP-induced genomic instability and the development of resistance.

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Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene

Cited by 38 publications

References 28 publications

Molecular Chaperone Hsp90 Regulates REV1-Mediated Mutagenesis

Molecular Chaperone Hsp90 Regulates REV1-Mediated Mutagenesis

Y-family DNA polymerases in mammalian cells

Human REV1 Modulates the Cytotoxicity and Mutagenicity of Cisplatin in Human Ovarian Carcinoma Cells

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