MECHANISMS OF MUTAGENESIS BY THE VINYL-CHLORIDE METABOLITE CHLOROACETALDEHYDE - EFFECT OF GENE-TARGETED INVITRO ADDUCTION OF M13 DNA ON DNA-TEMPLATE ACTIVITY INVIVO AND INVITRO

2-Chloroacetaldehyde (CAA), a metabolite of the carcinogenic industrial chemical vinyl chloride, reacts with single-stranded DNA to form the cyclic etheno lesions predominantly at adenine and cytosine. In both ethenoadenine and ethenocytosine, normal Watson-Crick hydrogen-bonding atoms are compromised. We have recently shown that CAA adduction leads to efficient mutagenesis in Escherichia coli predominantly at cytosines, and less efficiently at adenines. About 80% of the mutations at cytosines were C-to-T transitions, and the remainder were C-to-A transversions, a result similar to that of many noninstructional DNA lesions opposite which adenine residues are preferentially incorporated. It is widely believed that noninstructional lesions stop replication and depend on SOS functions for efficient mutagenesis. We have examined the effects of in vitro CAA adduction of the lacZα gene of phage M13AB28 on in vivo mutagenesis in SOS-(UV)-induced E. coli. CAA adduction was specifically directed to a part of the lacZ sequence within M13 replicative form DNA by a simple experimental strategy, and the DNA was transfected into appropriate unirradiated or UV-irradiated cells. Mutant progeny were defined by DNA sequencing. In parallel in vitro experiments, the effects of CAA adduction on DNA replication by E. coli DNA polymerase I large (Klenow) fragment were examined. Our data do not suggest a strong SOS dependence for mutagenesis at cytosine lesions. While adenine lesions remain much less mutagenic than cytosine lesions, mutation frequency at adenines is increased by SOS. SOS induction does not significantly alter the specificity of base changes at cytosines or adenines. The in vitro replication results show that these lesions create kinetic pause sites rather than absolute replication blocks. The simplest interpretation of the specificity of base changes is that the mechanisms of base incorporation opposite these lesions are analogous to those opposite the canonical noninstructional lesions. The high efficiency of mutagenesis opposite cytosine lesions without the aid of induced levels of SOS functions suggests that all DNA lesions lacking normal Watson-Crick base-pairing ability may nevertheless not block replication. In addition, the relatively nonmutagenic bypass of adenine lesions focuses attention on the need to understand mechanisms of error avoidance in the absence of normal base-pairing information. © 1990, American Chemical Society. All rights reserved.