Sequence-specific p53 gene damage by chloroacetaldehyde and its repair kinetics in Escherichia coli.
Oxidative stress and certain environmental carcinogens, e.g. vinyl chloride and its metabolite chloroacetaldehyde (CAA), introduce promutagenic exocyclic adducts into DNA, among them 1,N(6)-ethenoadenine (epsilonA), 3,N(4)-ethenocytosine (epsilonC) and N(2),3-ethenoguanine (epsilonG). We studied sequence-specific interaction of the vinyl-chloride metabolite CAA with human p53 gene exons 5-8, using DNA Polymerase Fingerprint Analysis (DPFA), and identified sites of the highest sensitivity. CAA-induced DNA damage was more extensive in p53 regions which revealed secondary structure perturbations, and were localized in regions of mutation hot-spots. These perturbations inhibited DNA synthesis on undamaged template. We also studied the repair kinetics of CAA-induced DNA lesions in E. coli at nucleotide resolution level. A plasmid bearing full length cDNA of human p53 gene was modified in vitro with 360 mM CAA and transformed into E. coli DH5alpha strain, in which the adaptive response system had been induced by MMS treatment before the cells were made competent. Following transformation, plasmids were re-isolated from transformed cultures 35, 40, 50 min and 1-24 h after transformation, and further subjected to LM-PCR, using ANPG, MUG and Fpg glycosylases to identify the sites of DNA damage. In adaptive response-induced E. coli cells the majority of DNA lesions recognized by ANPG glycosylase were removed from plasmid DNA within 35 min, while MUG glycosylase excised base modifications only within 50 min, both in a sequence-dependent manner. In non-adapted cells resolution of plasmid topological forms was perturbed, suggesting inhibition of one or more bacterial topoisomerases by unrepaired epsilon-adducts. We also observed delayed consequences of DNA modification with CAA, manifesting as secondary DNA breaks, which appeared 3 h after transformation of damaged DNA into E. coli, and were repaired after 24 h.
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland.
Paweł
P
Kowalczyk
Jarosław M
JM
Cieśla
Murat
M
Saparbaev
Jacques
J
Laval
Barbara
B
Tudek
D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D018736:DNA Adducts / Q000187:drug effects / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D005091:Exons / Q000235:genetics; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D016133:Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D014752:Vinyl Chloride / Q000737:chemistry / Q000494:pharmacology
D018736:DNA Adducts; D004269:DNA, Bacterial; C004656:chloroacetaldehyde; D004259:DNA-Directed DNA Polymerase; D000079:Acetaldehyde; D014752:Vinyl Chloride