DNA polymerase beta bypasses in vitro a single d(GpG)-cisplatin adduct placed on codon 13 of the HRAS gene.

Abstract

We have examined the capacity of calf thymus DNA polymerases a, f, 6, and e to perform in vitro translesion synthesis on a substrate containing a single d(GpG)-cisplatin adduct placed on codon 13 of the human HRAS gene. We found that DNA synthesis catalyzed by DNA polymerases ca, 6, and E was blocked at the base preceding the lesion Among the possible mechanisms of mutagenesis is error-prone replication by cellular DNA polymerases past a DNA lesion followed by fixation of the mutation during subsequent rounds of replication. In eukaryotic cells, it is still unclear whether the replicative DNA polymerases a, 8, and E can carry out translesion synthesis either alone or with the help of accessory proteins. Alternatively, a separate DNA polymerase may be required for this process. For example, genetic evidence in the yeast Saccharomyces cerevisiae indicates that a putative DNA polymerase, the product of the REV3 gene, may be involved in error-prone translesion synthesis but not in normal DNA replication (1). DNA polymerase (3 is one of the five mammalian polymerases identified to date and is believed to function primarily in the repair of damaged DNA (2). However, DNA polymerase a may also have a role in replicative synthesis, since the enzyme can substitute for DNA polymerase I during DNA replication in Escherichia coli (3), and it is required for the conversion of single-stranded M13 DNA to double-stranded DNA in Xenopus oocytes and in oocyte nuclear extracts (4). cis-Diamminedichloroplatinum(II) (cisplatin) is an anticancer agent widely used in the treatment of ovarian, testicular, head, and neck carcinomas (5). It is believed that this compound exerts its cytotoxic properties by forming stable lesions on DNA, primarily intrastrand cross-links at the N-7 positions of adjacent guanine bases [d(GpG)-cisplatin or Pt-d(GpG)] (6). Replicative bypass of cisplatin adducts has been described in bacteria (7,8) and in eukaryotic cells (9). Recent work in our laboratory has demonstrated that a single-stranded DNA The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.vector bearing a unique intrastrand bifunctional adduct Ptd(GpG) at codon 13 of the human protooncogene HRAS is replicated in simian COS-7 cells and that such translesion synthesis may be mutagenic (10).To our knowledge, the capacities of the major mammalian replication enzymes to bypass the Pt-d(GpG) lesion have not been compared. To address this question, we have investigated the ability of purified calf thymus DNA polymerases a, (3, 8, and s to catalyze in vitro the bypass synthesis of a single Pt-d(GpG) adduct placed on codon 13 of the human HRAS protooncogene, the same sequence used for our previous in vivo studies (10). Results show that only DNA polymerase 3 is capable of in vitro translesional synthesis and indicate that its ability to initiate DNA replication opposite the cisplatin adduct may ...

Enhanced expression and activity of DNA polymerase β in human ovarian tumor cells: impact on sensitivity towards antitumor agents

“…Our laboratory (Canitrot et al, 1998;Ho mann et al, 1995) and other groups (Vaisman and Chaney, 2000;Vaisman et al, 1999) have demonstrated the ability of one of the mammalian DNA polymerases, Pol b, to e ciently catalyze error-prone translesion synthesis in vitro across the major intrastrand cross-links at the N-7 positions of adjacent guanine bases. (Figure 3a).…”

“…An additional mechanism of resistance may be the consequence of an increased capacity of the cell to tolerate platinum-DNA lesions. We (Canitrot et al, 1998;Ho mann et al, 1995) and others (Vaisman and Chaney, 2000;Vaisman et al, 1999) have shown that puri®ed Pol b has the potential to e ciently catalyze error-prone translesion synthesis in vitro across the major intra-strand crosslinks at the N-7 positions of adjacent guanine bases. This mechanism has been proposed to be the primary mechanism that caused decreased cisplatin sensitivity in a series of cell lines, especially in the human ovarian carcinoma 2008/C13*5.25 cells where enhanced translesion synthesis of platinum lesions, ranging from 2 ± 5-fold compared to sensitive cell lines, has been observed (Johnson et al, 1997).…”

Specific Interaction of DNA Polymerase β and DNA Ligase I in a Multiprotein Base Excision Repair Complex from Bovine Testis

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Optical Nanocomposites Based on High Nanoparticles Concentration and Its Holographic Application

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