Abstract:In Saccharomyces cerevisiae, most mutations induced by a wide range of mutagens arise during translesion replication employing the REV1 gene product and DNA polymerase . As part of an effort to investigate mammalian mutagenic mechanisms, we have identified cDNA clones of the human homologs of the yeast REV genes and examined their function in UV mutagenesis. Previously, we described the isolation of a human homolog of yeast REV3, the catalytic subunit of pol , and here report the identification and sequence of… Show more
“…There was also no observed difference in sensitivity to UV radiation between the knockdown cell line and the parental controls. Diminished mutagenesis without a significant change in cell survival was similarly observed for a foreskin fibroblast cell line expressing REV1 antisense RNA and treated with UV radiation [60]. The greater relative effect on mutagenesis compared to survival is analogous to the situation in yeast, indicating alternative pathways to increase survival.…”
Section: Consequences Of Rev3l Rev1 and Rev7 Reduction In Human Cellmentioning
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.
“…There was also no observed difference in sensitivity to UV radiation between the knockdown cell line and the parental controls. Diminished mutagenesis without a significant change in cell survival was similarly observed for a foreskin fibroblast cell line expressing REV1 antisense RNA and treated with UV radiation [60]. The greater relative effect on mutagenesis compared to survival is analogous to the situation in yeast, indicating alternative pathways to increase survival.…”
Section: Consequences Of Rev3l Rev1 and Rev7 Reduction In Human Cellmentioning
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.
“…The Rev1 structure and functions appear to be highly conserved in higher eukaryotes. Experimental reduction of REV1 expression in cultured human cells results in a decrease in UV-induced mutagenesis [98]. Polη in yeast is encoded by RAD30, whose inactivation [99] or mutations in the corresponding mammalian xeroderma pigmentosum variant (XPV) gene [100,101] lead to an increased susceptibility to UV-induced DNA damage.…”
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...
“…Several studies using small interfering RNA on mammalian TLS polymerases have shown that Rev1 and Rev3, but not Pol , are clearly required in UV-induced mutagenesis (9,19,20,26,35). However, it is currently unknown what kind of mechanism is involved in the observed hypermutability of XP-V cells.…”
Section: Role For Pol In Prevention Of Uv-induced Mesenchymal Tumorsmentioning
DNA polymerase(Pol ) is the product of the Polh gene, which is responsible for the group variant of xeroderma pigmentosum, a rare inherited recessive disease which is characterized by susceptibility to sunlightinduced skin cancer. We recently reported in a study of Polh mutant mice that Pol is involved in the somatic hypermutation of immunoglobulin genes, but the cancer predisposition of Polh ؊/؊ mice has not been examined until very recently. Another translesion synthesis polymerase, Pol , a Pol paralog encoded by the Poli gene, is naturally deficient in the 129 mouse strain, and the function of Pol is enigmatic. Here, we generated Polh Poli doubledeficient mice and compared the tumor susceptibility of them with Polh-or Poli-deficient animals under the same genetic background. While Pol deficiency does not influence the UV sensitivity of mouse fibroblasts irrespective of Polh genotype, Polh Poli double-deficient mice show slightly earlier onset of skin tumor formation. Intriguingly, histological diagnosis after chronic treatment with UV light reveals that Pol deficiency leads to the formation of mesenchymal tumors, such as sarcomas, that are not observed in Polh ؊/؊ mice. These results suggest the involvement of the Pol and Pol proteins in UV-induced skin carcinogenesis.
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