Suppressible base substitution mutations induced by angelicin (isopsoralen) in the Escherichia coli lacI gene: implications for the mechanism of SOS mutagenesis

Abstract

Angelicin-plus near-UV-induced mutations were umuC dependent in Escherichia coli K-12. Angelicin, a monofunctional psoralen derivative, is believed to damage DNA almost exclusively at pyrimidine bases. To broaden our knowledge about the mutagenic specificity of SOS-dependent mutagens, we determined the mutational specificity of 233 suppressible lacI mutations induced by angelicin. More than 90% of the nonsense mutations arose via transversion substitutions. The three most frequently mutated sites were at A T base pairs and accounted for more than one-third of all induced nonsense mutations. The two hottest sites were at the only occurrences of the 5'-TATA-3' tetranucleotide in lacI, a sequence expected to be a preferred binding site for a psoralen. Both A T-to-T. A and A. T-to-C * G transversions were well induced by angelicin treatment, but the frequency of each transversion depended on the particular site. We also detected significant induction of transversion mutations at G-C sites. The induction of transversions by an SOS-dependent mutagen that generates lesions at pyrimidines supports the idea that DNA lesions influence the selection of bases that are incorporated via the process of SOS repair.The mutagenicity of DNA lesions that block DNA synthesis in Escherichia coli depends largely on inducible functions provided by the umuDC operon and the recA gene (21,29,43). Although the biochemical functions of the UmuDC and RecA products in SOS mutagenesis (43) are unknown, one important feature of the process involving these proteins has been deduced: mutations generated via SOS mutagenesis occur at sites of DNA damage (9,14,28). Replication bypass of lesions on a template strand is the most straightforward explanation for the targeting of mutations to sites of DNA damage (16).The base substitution specificities of several different SOS-dependent mutagens were determined by employing the E. coli lacI nonsense system (27,28). All but two of the mutagens analyzed by this system, UV and neocarzinostatin, were agents which damage DNA preferentially at purines (summarized in reference 9). To broaden the base of knowledge about base substitution mutagenesis via the SOS response, we decided to examine the mutagenic specificity of an additional SOS-dependent mutagen that preferentially damages DNA at pyrimidines. Here we present the spectrum of lacI nonsense mutations induced by angelicin. Angelicin is a monofunctional psoralen that engages in specific photochemical reactions with pyrimidine bases in DNA after irradiation with light in the near-UV range (2, 6). The major products of reactions between psoralens and DNA are adducts linked by a cyclobutane ring to the 5-6 position of pyrimidines; these adducts block DNA replication in vitro (31,32). We show that mutation induction by angelicin treatment is umuC dependent in E. coli and evaluate the distribution of 233 angelicin-induced lacI nonsense mutations. Our analysis leads us to suggest that angelicin adducts to pyrimidines are mutagenic lesions that induce transversio...

Induction of base substitution mutations by aflatoxin B1 is mucAB dependent in Escherichia coli

“…The bacterial strains used are E. coli K-12 derivatives defective for the uvrABC excision repair system (20). EE270 is araA(gpt-1ac-pro) thi uvrB5 chlA::TnJO/F' lac+ proB+ (35), and PF203 is ara A(gpt-lacpro) thi uvrB5 Ahis-SIF' 400 hisOJ242 hisG46 gnd: :TnJO rfb+ (see reference 16 for related constructions). PF501 is AB1885 but recA441 sulAJi (9,17 (17,24).…”

8-Methoxypsoralen induced mutations are highly targeted at crosslinkable sites of photoaddition on the non-transcribed strand of a mammalian chromosomal gene.

“…In other bacterial and mammalian systems, psoralen derivatives also induce mainly point mutations primarily at A:T base pairs, although the T:A -A:T transversions may not be the preferred event (Piette et al, 1985;Miller and Eisenstadt, 1987;Yatagai et al, 1987). In the lacI gene of E. coli, 65% of the 8-MOPinduced mutations occurred at A:T base pairs (Yatagai et al, 1987).…”

“…In the lacI gene of E. coli, 65% of the 8-MOPinduced mutations occurred at A:T base pairs (Yatagai et al, 1987). In the same gene, hotspots of mutation induced by angelicin occur at T residues within a repeated TA context (Miller and Eisenstadt, 1987), which are strong sites for angelicin photobinding (Boyer et al, 1988;Miolo et al, 1989). In addition, all seven HPRT-4'-hydroxymethyl-4,5', 8-trimethylpsoralen (HMT)-induced base substitution mutations isolated from mouse cells were recovered at A:T base pairs, four within an ATA/TAT context (Piette, 1992).…”

“…In sharp contrast to the APRT locus, a significant proportion of mutations in bacterial genes, as well as in an extrachromosomal shuttle vector propagated in mammalian cells (Bredberg and Nachmansson, 1987;Miller and Eisenstadt, 1987;Yatagai et al, 1987), were recovered at G:C base pairs, where psoralen photoadducts are extremely rare. This may reflect differences in replisome and/or repairosome structure and function with respect to processing of psoralen-induced damage in endogenous mammalian genes versus shuttle vectors or bacteria.…”

MONOFUNCTIONAL ANGULAR FUROCOUMARINS: SEQUENCE SPECIFICITY IN DNA HOTOBINDING OF 6,4,4′‐TRIMETHYLANGELICIN and OTHER ANGELICINS

“…In this study, the DNA substrates for TMA photoreaction were parts of the lac I gene of Escherichia coli, a target gene often used for mutagenesis studies (Miller, 1982;Yatagai and Glickman, 1986). Indeed, the specificity of mutations induced by 8-MOP and angelicin plus UVA has already been analyzed in this gene (Miller and Eisenstadt, 1987;Yatagai et al, 1987).…”