Localization of chloroacetaldehyde-induced DNA damage in human p53 gene by DNA polymerase fingerprint analysis.
Chloroacetaldehyde (CAA) reacts with DNA bases, forming hydroxyethano derivatives of different stability, which are subsequently converted into etheno (epsilon) adducts: epsilon A, epsilon C, epsilon G. DNA polymerase fingerprint analysis was used to study the distribution of CAA-induced modifications in the p53 sequence. A plasmid bearing cDNA containing the human p53 gene was reacted in vitro with CAA, then dehydrated for conversion of hydroxyethano into etheno adducts, and primer extension by T7 DNA polymerase in the presence of four dNTPs was performed. The DNA repair enzymes methylpurine-DNA glycosylase and Escherichia coli exonuclease III were used to convert epsilon A residues in the template into DNA strand breaks, which enabled precise localization of the epsilon A residues within the p53 gene. Hydroxyethano derivatives of adenine and cytosine in a template blocked T7 DNA polymerase and caused premature chain termination opposite adenine or one base before cytosine. After dehydration, both epsilon A and epsilon C were much more easily by-passed by T7 DNA polymerase. Formation of epsilon G was identified as 'stop bands' one base before guanine residues. Modification of cytosine and guanine was additionally recognized by weakening or disappearance of non-specific stops on an undamaged template, probably due to steric hindrance by the tertiary DNA structure for polymerase. Etheno adduction of cytosine and guanine relaxed the compact DNA structure and enabled DNA polymerase to by-pass. In exons 5-8 of p53, 143 out of 500 sites appeared to be damaged by CAA, with four particularly densely modified regions between codons 135-147, 218-222, 234-255 and 284-292. The pattern of modification followed the pattern of p53 mutations found in vinyl chloride-associated liver angiosarcomas in humans and rats, but only in regions that showed 100% homology with the human sequence. The factors that influence DNA damage and induction of mutations in the p53 gene by CAA and vinyl chloride are discussed.
Tudek
B
B
Polish Academy of Sciences, Institute of Biochemistry and Biophysics, Warszawa, Poland.
Kowalczyk
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P
Cieśla
J M
JM
D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000633:toxicity; D000225:Adenine / Q000378:metabolism; D000818:Animals; D003596:Cytosine / Q000378:metabolism; D004249:DNA Damage; D016172:DNA Fingerprinting / Q000379:methods*; D004260:DNA Repair; D005091:Exons; D016158:Genes, p53 / Q000187:drug effects*; D006147:Guanine / Q000378:metabolism; D006394:Hemangiosarcoma / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D051381:Rats; D017422:Sequence Analysis, DNA / Q000379:methods
D006147:Guanine; D003596:Cytosine; C004656:chloroacetaldehyde; D000079:Acetaldehyde; D000225:Adenine