Comparison of the sequence specificity of cis-diamminedichloroplatinum (II) damage in guanine- and 7-deazaguanine-containing DNA.
The N7 of guanine is thought to be the primary target for adduct and crosslink formation between cisplatin and DNA. However, reactive sites in DNA other than the N7 of guanine may also participate in the formation of adducts with cisplatin. The possibility that these interactions arise and form DNA polymerase blocking lesions was investigated by primer extension reactions with Taq DNA polymerase. To differentiate between damage produced at relatively weak sites from those formed at the N7 of guanine, a modified DNA template was synthesised with the N7 of guanine replaced with a carbon atom. This was achieved in a PCR designed to incorporate 7-deazaguanine instead of normal guanine. The sequence specificity of cisplatin damage in the modified and unmodified DNA substrates was compared (after linear amplification) by DNA sequencing gel analysis. For concentrations of cisplatin (1 to 5 microM) that induce blocking lesions in normal DNA, no significant damage was observed in the modified DNA. This confirmed that the N7 of guanine is the major site of adduct formation in normal DNA. At higher concentrations of cisplatin (50 microM and 100 microM), lesions were found at AA dinucleotides and other novel sites in the modified DNA. These results indicate that the N7 of guanine is not required in the formation of some cisplatin adducts.
Cairns
M J
MJ
School of Biochemistry and Molecular Genetics, University of New South Wales, Kensington, Australia.
Murray
V
V
D001483:Base Sequence; D002945:Cisplatin / Q000633:toxicity*; D004247:DNA / Q000737:chemistry / Q000187:drug effects* / Q000302:isolation & purification; D004249:DNA Damage; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006147:Guanine / Q000031:analogs & derivatives*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D019914:Taq Polymerase
D017931:DNA Primers; D006147:Guanine; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; C066856:7-deazaguanine; D002945:Cisplatin