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 damage. Most DNA damage is noninstructive in the sense that the causative chemical modification either destroys the template information or renders it inaccessible to the DNA polymerase. Noninstructive adducts possess high genotoxicity because they stop DNA replication. Replication past noninstructive adducts is thought to depend on induced functions in addition to the regular replication machinery. In Escherichia coli, noninstructive DNA damage leads to induction of the SOS regulon, which in turn is thought to provide the inducible functions required for replicative bypass of the lesion. Because of the absence of accessible template 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 vinyl chloride and urethane, is a highly mutagenic, noninstructive lesion on the basis of its template characteristics in vivo and in vitro. However, mutagenesis at epsilonC does not require SOS functions, as evidenced by efficient mutagenesis in recA-deleted E. coli. Even though efficient mutagenesis in recA-deleted cells shows a lack of SOS dependence, the question remains whether SOS induction can modulate mutagenesis opposite epsilonC. To examine the possible contribution of SOS functions to mutagenesis at epsilonC, we constructed an M13 duplex circular DNA molecule containing an epsilonC residue at a unique site. The construct was transfected into nonirradiated or UV-irradiated E. coli. The frequency as well as specificity of the mutations induced under a number of conditions was determined by using a multiplex DNA sequencing technology. Without prior UV irradiation, approximately one-third of the progeny is mutant, the majority of mutations being C-->T transitions. Prior UV irradiation of wild-type host cells results in a significant increase in mutagenesis with most of the increase accounted for by an increase in C-->A transversions. Surprisingly, essentially identical effects were observed in irradiated recA-deleted cells as well as in umuC-deficient cells, suggesting that the observed UV modulation of mutagenesis is independent of the SOS pathway. These observations suggest the existence of a recA-independent UV-inducible mutagenic mechanism in E. coli.