Mechanisms of mutagenesis by exocyclic DNA adducts. Transfection of M13 viral DNA bearing a site-specific adduct shows that ethenocytosine is a highly efficient RecA-independent mutagenic noninstructional lesion.
It is widely accepted that mutagenic DNA lesions fall into two categories: mispairing lesions hydrogen bond with an incorrect incoming base, generally do not stop replication, and possess high mutagenic efficiency without any requirement for induced functions; noninstructional lesions lack accessible template information, act as strong blocks to DNA replication (and are therefore toxic), and their mutagenic effects are SOS-dependent. Our recent results show that ethenocytosine (epsilon C), a noninstructional exocyclic DNA lesion induced by vinyl chloride, may have unusual mutagenic properties. To obtain more definitive experimental evidence for the observed effects, we have introduced a single epsilon C residue at a specific site of coliphage M13AB28 replicative form DNA by a "single-stranded linker-ligation" technique. The resulting DNA was purified and transfected into appropriate recA+ or recA- Escherichia coli host cells. The effect of epsilon C on survival was determined from transfection efficiency. Both the frequency and specificity of mutations induced by epsilon C were determined by direct sequence analysis of randomly picked progeny phage plaques. The results indicated that epsilon C has little effect on the survival of M13 DNA. Approximately 30% of the progeny phage obtained by transfecting epsilon C DNA had a base substitution mutation precisely at the lesion site. No such mutations were observed in progeny plaques obtained by transfecting the control DNA construct. All epsilon C-induced mutations were either C-to-T transitions or C-to-A transversions. Neither survival nor mutagenic efficiency was significantly affected in recA- host cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Palejwala
V A
VA
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103-2714.
Simha
D
D
Humayun
M Z
MZ
D000477:Alkylating Agents; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics* / Q000254:growth & development; D003596:Cytosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D004270:DNA, Circular / Q000138:chemical synthesis / Q000187:drug effects / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000138:chemical synthesis / Q000302:isolation & purification; D004279:DNA, Viral / Q000138:chemical synthesis / Q000737:chemistry / Q000187:drug effects*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011938:Rec A Recombinases / Q000235:genetics*; D014162:Transfection
C018289:3,N(4)-ethenocytosine; D000477:Alkylating Agents; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D003596:Cytosine; D011938:Rec A Recombinases