MECHANISMS OF MUTAGENESIS BY EXOCYCLIC DNA ADDUCTS - CONSTRUCTION AND INVITRO TEMPLATE CHARACTERISTICS OF AN OLIGONUCLEOTIDE BEARING A SINGLE SITE-SPECIFIC ETHENOCYTOSINE

By using a gene-targeted random DNA adduction approach, we have recently shown that chloroacetaldehyde, a metabolite of vinyl chloride, induces mutations predominantly at cytosines under conditions in which both ethenoadenine (epsilon-A) and ethenocytosine (epsilon-C) are formed. Although the observed mutational specificity of epsilon-C suggested that it was a noninstructional lesion, the high efficiency of mutagenesis and an apparent lack of SOS dependence were reminiscent of mispairing lesions. To obtain more direct evidence showing that epsilon-C has properties of a noninstructional mutagenic lesion, we have examined the in vitro template properties of a single epsilon-C residue at a unique position in a synthetic oligonucleotide. The oligonucleotide was constructed by use of the following steps: (a) in vitro treatment of the pentameric oligodeoxyribonucleotide TTCTT with chloroacetaldehyde to convert the central cytosine to ethenocytosine; (b) purification and characterization of TT-epsilon-CTT; and (c) ligation of purified TT-epsilon-CTT to two decamers to create a 25 nt long oligodeoxyribonucleotide with a centrally located epsilon-C residue. The template characteristics of epsilon-C were examined by the annealing of end-labeled primers to the purified epsilon-C-containing oligonucleotide and primer elongation by Escherichia coli DNA polymerase I in the presence of one or more nucleotide precursors. The elongation products were analyzed by high-resolution gel electrophoresis followed by autoradiography and quantitated by computing densitometry. The results indicated the following: (a) at low nucleotide precursor concentrations at which guanine incorporation can be readily demonstrated opposite normal cytosine, no detectable base incorporation occurs opposite epsilon-C; (b) base incorporation opposite epsilon-C can be detected in the presence of relatively high concentrations of nucleotide precursors; adenine is preferentially incorporated, followed by thymine, whereas guanine and cytosine are poorly incorporated. These results establish that epsilon-C lesions display the in vitro template properties expected of noninstructional lesions. However, in this experimental system, continued synthesis beyond the epsilon-C site is most efficient when the incorporated base is a thymine.