Bypass Replication of the Cisplatin-d(GpG) Lesion by Calf Thymus DNA Polymerase β and Human Immunodeficiency Virus Type I Reverse Transcriptase Is Highly Mutagenic

Abstract

Eukaryotic DNA polymerase ␤ and the reverse transcriptases are the most inaccurate of the known DNA polymerases. We report here mutagenic replication in vitro past intrastrand N(7)G-N(7)G chelates of the cisdiamminedichloroplatinum(II), the major DNA adduct of the antitumor agent cisplatin by calf thymus DNA polymerase ␤ and human immunodeficiency virus type I reverse transcriptase (42% and 26% mutations, respectively). The most frequent modifications generated by both enzymes were one-base frameshift deletions. Only one mutational hot spot opposite the platinated guanines was observed with human immunodeficiency virus type I reverse transcriptase, while two hot spots were generated by DNA polymerase ␤, one at the base situated 5 to the lesion and the other situated 4 -6 nucleotides 5 to the adduct. An unusual mutagenic event, tandem replication of a 12-base pair sequence, was observed with DNA polymerase ␤. The mutational spectra of the two DNA polymerases suggest that template slippage occurred with higher frequency in the presence of the more distributive DNA polymerase ␤.The molecular mechanisms of mutagenesis are not well understood. One general pathway is thought to involve errorprone replication past unrepaired DNA damage. Bulky DNA lesions can result from cellular exposure to agents such as UV light or a variety of chemical carcinogens. Studies performed in vivo indicate that mammalian cells possess the ability to circumvent blocks to DNA synthesis and are able to replicate in the presence of these lesions (1-4). In vitro, it has been demonstrated that a number of purified cellular DNA polymerases can replicate through bulky adducts. For example, calf thymus DNA polymerase ␦ has been shown to replicate past cis-synand trans-syn-cyclobutane-thymine dimers in the presence of proliferating cell nuclear antigen (5). We have recently shown that an intrastrand cross-link formed by the anticancer and mutagenic agent cis-diamminedichloroplatinum(II) (cisplatin) blocked calf thymus DNA polymerases ␣, ␦, and ⑀, while DNA polymerase ␤ is capable of replicating past the lesion (6). This Pt-d(GpG) 1 adduct was placed at the N-7 position of the two adjacent guanine bases of codon 13 of the human HRAS gene. DNA polymerase ␤, the structurally simplest of the five known mammalian DNA polymerases, is believed to function primarily in the repair of damaged DNA. It is a monomeric protein of 335 amino acids (39 kDa) that lacks exonuclease activities and works in a distributive manner, although it is capable of processive gap-filling synthesis on single-stranded DNA templates for up to 6 nucleotides (7). DNA polymerase ␤ appears to be the least accurate of the eukaryotic DNA polymerases in replicating undamaged DNA in vitro (8, 9). Human immunodeficiency virus reverse transcriptase (HIV-1 RT) also catalyzes highly error-prone replication (10 -14) and shows structural homology with DNA polymerase ␤ (15).The mutagenic consequences of the replicative bypass of a single Pt-d(GpG) adduct in vitro are unknown. We compare here the...

Modulation of Cellular Response to Cisplatin by a Novel Inhibitor of DNA Polymerase β

“…It is noteworthy that the presence of the adduct changes the synthesis from distributive to processive in nature. We discussed this aspect in previous articles (Hoffmann et al, 1995(Hoffmann et al, , 1996, explaining that when the damage is present, the enzyme dissociates frequently from the adducted bases, resulting in more availability of Pol ␤ to reinitiate primer extension and to extend most of the [3Ј-OH] termini to Fig. 3.…”

Effect of Loss of DNA Mismatch Repair on Development of Topotecan-, Gemcitabine-, and Paclitaxel-Resistant Variants after Exposure to Cisplatin

“…The most abundant lesions produced in DNA are intrastrand cross-links between the N7 atoms of adjacent purines; 65% of adducts are in GpG sequences, 25% are in ApG sequences, and 6% are in GpNpG sequences (Eastman, 1986). Such adducts halt the progress of DNA polymerases when they are present in the lagging strand, but mutagenic translesional bypass occurs at a significant level on the leading strand and results in the misincorporation of nucleotides opposite the intrastrand adducts (Hoffmann et al, 1996). Intrastrand guanine-guanine adducts themselves and compound lesions consisting of the adduct and a misincorporated base on the opposite strand are recognized and processed by the MMR system (Duckett et al, 1996;Yamada et al, 1997).…”

Structure of DNA polymerase β with a benzo[ c ]phenanthrene diol epoxide-adducted template exhibits mutagenic features

“…Over-expression of Pol ␤ can result in decreased sensitivity to agents that generate bulky DNA adducts, such as cisplatin (30), or UV radiation (31). In vitro assays demonstrate the ability of Pol ␤ to bypass UVinduced (31) and cisplatin-induced DNA lesions (32).…”