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      Involvement of Vertebrate Polκ in Translesion DNA Synthesis across DNA Monoalkylation Damage

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          Abstract

          DNA lesions that escape excision repair pathways can cause arrested DNA replication. This replication block can be processed by translesion DNA synthesis (TLS), which is carried out by a number of specialized DNA polymerases. A sequential lesion bypass model has been proposed; one of the lesion-specific polymerases inserts nucleotide(s) opposite the damaged template, followed by extension from the inserted nucleotide by the same or another polymerase. Polzeta and Polkappa have been proposed as candidates for executing the extension step in eukaryotic cells. We previously disrupted separately Rev3, the catalytic subunit of Polzeta, and Polkappa in chicken B lymphocyte DT40 cells. We found that each cell line showed significant UV sensitivity, implying that both contribute to UV radiation damage repair. In the present studies we generated REV3(-/-)POLK(/-) double knock-out cells to determine whether they participate in the same or different pathways. The double mutant was viable and proliferated with the same kinetics as parental REV3(-/-) cells. The cells showed the same sensitivity as REV3(-/-) cells to UV, ionizing radiation, and chemical cross-linking agents. In contrast, they were more sensitive than REV3(-/-) cells to monofunctional alkylating agents, even though POLK(/-) cells barely exhibited increased sensitivity to those. Moreover Polk-deficient mouse embryonic stem and fibroblast cells, both of which have previously been shown to be sensitive to UV radiation, also showed moderate sensitivity to methyl methanesulfonate, a monofunctional alkylating agent. These data imply that Polkappa has a function in TLS past alkylated base adducts as well as UV radiation DNA damage in vertebrates.

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          Most cited references28

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          Mechanisms of accurate translesion synthesis by human DNA polymerase eta.

          The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta (pol eta), which is involved in the replication of damaged DNA. Pol eta catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol eta can catalyze translesion synthesis past an abasic (AP) site analog, N-2-acetylaminofluorene (AAF)-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Pol eta preferentially incorporated dAMP and dGMP opposite AP, and dCMP opposite AAF-G and cisplatin-GG, but other nucleotides were also incorporated opposite these lesions. However, after incorporating an incorrect nucleotide opposite a lesion, pol eta could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol eta could continue chain elongation, whereas pol alpha could not. Thus, the fidelity of translesion synthesis by human pol eta relies not only on the ability of this enzyme to incorporate the correct nucleotide opposite a lesion, but also on its ability to elongate only DNA chains that have a correctly incorporated nucleotide opposite a lesion.
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            Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis.

            Pol kappa and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass [translesion DNA synthesis (TLS)]. We demonstrate that mouse Rev1 protein physically associates with Pol kappa. We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Pol zeta) and with two other Y-family polymerases, Pol iota and Pol eta. Mouse Pol kappa, Rev7, Pol iota and Pol eta each bind to the same approximately 100 amino acid C-terminal region of Rev1. Furthermore, Rev7 competes directly with Pol kappa for binding to the Rev1 C-terminus. Notwithstanding the physical interaction between Rev1 and Pol kappa, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini. Our observations suggest that Rev1 plays a role(s) in mediating protein-protein interactions among DNA polymerases required for TLS. The precise function(s) of these interactions during TLS remains to be determined.
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              Translesion DNA synthesis in eukaryotes: a one- or two-polymerase affair.

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                Author and article information

                Journal
                Journal of Biological Chemistry
                J. Biol. Chem.
                American Society for Biochemistry & Molecular Biology (ASBMB)
                0021-9258
                1083-351X
                January 20 2006
                January 27 2006
                January 27 2006
                November 23 2005
                : 281
                : 4
                : 2000-2004
                Article
                10.1074/jbc.M506153200
                16308320
                592b5356-51b1-4183-aeec-19ef45c4ef0f
                © 2005
                History

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