Mutational specificity of 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea in the Escherichia coli lacl gene of O⁶‐alkylguanine‐DNA alkyltransferase‐proficient and ‐deficient strains

Abstract

Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the lacl gene of Escherichia coli were recovered from bacteria proficient (Ogt+ Ada+) and deficient (Ogt- Ada-) in O6-alkylguanine-DNA alkyltransferase activity. A CCNU dose of 1 mM was selected for DNA sequence analysis. A total of 245 induced mutations were characterized. The mutations were almost exclusively (95%) GC-->AT transitions, indicating that CCNU-induced mutations arose in bacteria primarily from misreplication of O6-chloroethylguanine, in total agreement with results obtained for monofunctional alkylating agents. The distribution of CCNU-induced GC-->AT mutations was significantly altered by the presence of DNA alkyltransferase activity (P = 0.01). In the Ogt+ Ada+ mutational spectrum, guanines flanked on both sides by A:T base-pairs were on average 2.8 times more likely to mutate than those flanked by G:C base-pairs on at least one side. This bias disappeared in the Ogt- Ada- genetic background, thereby providing evidence that O6-chloroethylated guanines adjacent to G:C base-pairs are better targets for bacterial alkyltransferase than those not adjacent to G:C base-pairs. We recently reported a similar bias for ethyl methanesulfonate, strengthening the idea that CCNU is acting as a simple ethylating compound. In summary, this paper presents for the first time evidence that DNA repair by O6-alkylguanine-DNA alkyltransferases plays a major role in removing lesions responsible for GC-->AT transitions induced by CCNU, influencing their ultimate distribution with respect to sequence context.

Determining mutational fingerprints at the human p53 locus with a yeast functional assay: a new tool for molecular epidemiology

“…At both loci, the incidence and the location of GC4AT transitions are consistent with the hypothesis that O6-alkylguanine is the main determinant of CCNU mutagenicity. Alkylnitrosoureas preferentially cause GC4AT transitions at Gs preceded by a Pu residue in E. coli (Jurado et al, 1995;RoldaÁ n-Arjona et al, 1994;Richardson et al, 1987;Burns et al, 1988) and in eukaryotes (Inga et al, 1995;Kunz and Mis, 1989;Minnick et al, 1992;Moriwaki et al, 1991;Palombo et al, 1992). The 5'-¯anking purine eect has been explained as the consequence of a structural in¯uence on the initial formation of O6-alkylguanine, which may be further enhanced by alkylation speci®c repair (Horsfall et al, 1990).…”

Mutation spectra analysis suggests that N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea-induced lesions are subject to transcription-coupled repair in Escherichia coli

“…Differences in survival and mutability between uvr + and uvrbacteria [27][28][29] as well as between ATase + and ATasebacteria have been reported [9,29]. On the other hand, Kohda et al [30] found that uvrAand uvrA + strains treated with chloroethyl-nitrosourea showed no differences in survival and mutability.…”

“…Indeed, all strains we examined had similar incidences of GC→AT transitions ( Table 2) that occurred in similar sequence contexts (more than 96% were preceded 5´ by a purine) ( Table 4). It is known that alkylnitrosoureas preferentially cause GC→AT transitions at Gs preceded by a purine residue in E. coli [9,32,33] and in eukaryotes [1,34,35]. The 5´-flanking purine effect has been explained as the consequence of structural influence on the initial formation of O 6 -chloroethylguanine that may be further enhanced by sequence-specific repair [36].…”

“…Very recently, Jurado et al [9] determined the CCNU spectra at the lacI locus in uvrBada + ogt + and uvrBadaogtstrains. They found that in both spectra, more than 95% of mutations were GC→AT transitions.…”

“…G-C cross-links have been best correlated with cytotoxicity in cell-culture studies, suggesting that the cytotoxic effect of CCNU on cancer cells is mediated by this type of damage [5]. CCNU is mutagenic in prokaryotes [6][7][8][9] and eukaryotes [10,11], but the premutational lesions remain poorly defined. Different DNA repair pathways, among which include the O 6 -alkylguanine-DNA alkyltransferase (ATase), the nucleotide excision repair, and the N3-alkyladenine DNA glycosylase activities, have been reported to protect prokaryotes and eukaryotes from the lethal and mutagenic effects of haloethylnitrosourea-induced lesions [3,12,13].…”