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

Biochemistry. 1991 Sep 10;30(36):8736-43. doi: 10.1021/bi00100a004.


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)

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkylating Agents
  • Base Sequence
  • Coliphages / genetics*
  • Coliphages / growth & development
  • Cytosine / analogs & derivatives*
  • Cytosine / pharmacology
  • DNA Replication / drug effects
  • DNA, Circular / chemical synthesis
  • DNA, Circular / drug effects
  • DNA, Circular / isolation & purification
  • DNA, Single-Stranded / chemical synthesis
  • DNA, Single-Stranded / isolation & purification
  • DNA, Viral / chemical synthesis
  • DNA, Viral / chemistry
  • DNA, Viral / drug effects*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Rec A Recombinases / genetics*
  • Transfection*


  • 3,N(4)-ethenocytosine
  • Alkylating Agents
  • DNA, Circular
  • DNA, Single-Stranded
  • DNA, Viral
  • Cytosine
  • Rec A Recombinases