Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli.

Abstract

The mutation spectrum induced by the widely used antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) showed that cisDDP{d(ApG)} adducts, although they account for only 25% of the lesions formed, are =5 times more mutagenic than the major GG adduct. We report the construction of vectors bearing a single cisDDP{d(ApG)} lesion and their use in mutagenesis experiments in Escherichia coli. The mutagenic processing of the lesion is found to depend strictly on induction of the SOS system of the bacterial host cells. In SOS-induced cells, mutation frequencies of 1-2% were detected. All these mutations are targeted to the 5' base of the adduct. Single A -+ T transversions are mainly observed (80%), whereas A -* G transitions account for 10% of the total mutations. Tandem base-pair substitutions involving the adenine residue and the thymine residue immediately 5' to the adduct occur at a comparable frequency (10%). No selective loss of the strand bearing the platinum adduct was seen, suggesting that, in vivo, cisDDP{d(ApG)} adducts are not blocking lesions. The high mutation specificity of cisDDP{d(ApG)}-induced mutagenesis is discussed in relation to structural data.During the past decade, progress in molecular biology allowed the development of strategies for elucidating the molecular mechanisms involved in processing the damages introduced in DNA by physical or chemical agents. A general procedure is to determine the forward mutation spectrum induced by a particular agent on a target DNA sequence (1). This approach allows definition of the general characteristics of the mutagenic processes, such as their genetic requirements and their specificity. However, these strategies are not sensitive enough to measure the relative contribution of different adducts in repair or mutagenesis events. This problem has been overcome by the use of synthetic oligodeoxyribonucleotides containing defined adducts located at precise sites (for review, see ref.2). When these adducts are introduced in a replicative vector and transfected into cells, several biological endpoints can be monitored: toxicity (3, 4), mutation efficiency and specificity (3-6), effect of the repair genotype of the host (7), and effect of surrounding sequences (3, 6). Furthermore, such modified oligonucleotides became useful tools for studying the structure of the DNA helix in the vicinity of the adduct (8-13) and precise biochemistry of repair (14-16).cis-Diamminedichloroplatinum(II) (cisDDP) is a widely used antitumor drug that binds to DNA, its main cellular target, by preferentially forming intrastrand crosslinks between the N7 atoms of adjacent purines (17)(18)(19). At low modification levels, the main adducts result from the chelation of two adjacent guanines [cisDDP{d(GpG)}], 65% of total lesions), whereas 25% of the adducts arise at 5' d(ApG) sequences. d(GpHpG) adduct (H = A, C, or T) and interstrand crosslinks altogether represent <10% of the total lesions (20-22).We have described the spectrum of mutations induced by cisDDP in Escherichia c...

Recognition and Repair of Compound DNA Lesions (Base Damage and Mismatch) by Human Mismatch Repair and Excision Repair Systems

“…The results presented so far suggest that compound lesions are more mutagenic than simple lesions because they are removed more efficiently by excision repair and recognized less efficiently by the mismatch repair system. Most cisplatincaused mutations at GG sequences are G-C to A-T transitions at the 5Ј guanine (3,6), consistent with our use of a G-T mismatch in the cisplatin 1,2-d(GpG) compound lesion (Fig. 2).…”

“…The general nucleotide excision repair (excision repair) system (8) not only is the sole repair pathway for bulky lesions such as thymine dimers (TϽϾT) and cisplatin-guanine adducts but also repairs a wide variety of nonbulky lesions such as O 6 -methylguanine (O 6 -meG) at physiologically relevant rates (52). Thus, it appears that both repair systems recognize many dissimilar non-B DNA forms rather than a specific lesion structure.…”

Fork-like DNA templates support bypass replication of lesions that block DNA synthesis on single-stranded templates

“…The mutation specificity of the two adducts is also in agreement with this drastic difference in the conformational change induced in the DNA structure. Indeed, platinum adducts induce base pair substitutions (17)(18)(19) while AAF adducts essentially induce frameshift mutations (20)(21)(22).…”

Induction of frameshift and base pair substitution mutations by the major DNA adduct of the endogenous carcinogen malondialdehyde

“…3). The cause of these untargeted mutations is unclear, although the ability of one DNA adduct to cause more than one mutation, even several bases away, has been observed by others (35)(36)(37)(38). M 1 G may introduce an area of instability in DNA that could interfere with faithful replication beyond the adduct, causing multiple mistakes to be made by DNA polymerases.…”