UV IRRADIATION OF ESCHERICHIA-COLI MODULATES MUTAGENESIS AT A SITE-SPECIFIC ETHENOCYTOSINE RESIDUE ON M13 DNA - EVIDENCE FOR AN INDUCIBLE RECA-INDEPENDENT EFFECT

Mutagenic action of chemical and physical mutagens is mediated through DNA damage and subsequent misreplication at sites of unrepaired damag e. Most DNA damage is noninstructive in the sense that the causative c hemical modification either destroys the template information or rende rs it inaccessible to the DNA polymerase. Noninstructive adducts posse ss high genotoxicity because they stop DNA replication. Replication pa st noninstructive adducts is thought to depend on induced functions in addition to the regular replication machinery. In Escherichia coli, n oninstructive DNA damage leads to induction of the SOS regulon, which in turn is thought to provide the inducible functions required for rep licative bypass of the lesion. Because of the absence of accessible te mplate instruction, base incorporation opposite noninstructive lesions is inherently error-prone and results in mutagenesis. Ethenocytosine (epsilonC), an exocyclic DNA lesion induced by carcinogens such as vin yl chloride and urethane, is a highly mutagenic, noninstructive lesion on the basis of its template characteristics in vivo and in vitro. Ho wever, mutagenesis at epsilonC does not require SOS functions, as evid enced by efficient mutagenesis in recA-deleted E. coli. Even though ef ficient mutagenesis in recA-deleted cells shows a lack of SOS dependen ce, the question remains whether SOS induction can modulate mutagenesi s opposite epsilonC. To examine the possible contribution of SOS funct ions to mutagenesis at epsilonC, we constructed an M13 duplex circular DNA molecule containing an epsilonC residue at a unique site. The con struct was transfected into nonirradiated or UV-irradiated E. coli. Th e frequency as well as specificity of the mutations induced under a nu mber of conditions was determined by using a multiplex DNA sequencing technology. Without prior UV irradiation, approximately one-third of t he progeny is mutant, the majority of mutations being C-->T transition s. Prior UV irradiation of wild-type host cells results in a significa nt increase in mutagenesis with most of the increase accounted for by an increase in C-->A transversions. Surprisingly, essentially identica l effects were observed in irradiated recA-deleted cells as well as in umuC-deficient cells, suggesting that the observed UV modulation of m utagenesis is independent of the SOS pathway. These observations sugge st the existence of a recA-independent UV-inducible mutagenic mechanis m in E. coli.