Mechanisms of mutagenesis by exocyclic DNA adducts. Construction and in vitro 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Simha
D
D
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103-2714.
Palejwala
V A
VA
Humayun
M Z
MZ
D000477:Alkylating Agents; D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives*; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D004249:DNA Damage; D004256:DNA Polymerase I / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D013698:Templates, Genetic
C018289:3,N(4)-ethenocytosine; D000477:Alkylating Agents; D009838:Oligodeoxyribonucleotides; D003596:Cytosine; D004247:DNA; D004256:DNA Polymerase I