In vivo formation and in vitro replication of a guanine-thymine intrastrand cross-link lesion.
G[8-5m]T, a guanine-thymine intrastrand cross-link lesion where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon, was previously shown to form in an aqueous solution of duplex DNA upon exposure to gamma- or X-rays and in calf thymus DNA treated with Fenton reagents. Here, we employed LC-MS/MS and demonstrated for the first time that this lesion could be induced in a dose-dependent manner in human Hela-S3 cells upon exposure to gamma-rays. We further carried out in vitro replication studies on a substrate containing a site-specifically incorporated G[8-5m]T, and our results showed that the Klenow fragment of Escherichia coli DNA polymerase I stopped synthesis mostly after incorporating the correct nucleotide dAMP opposite the 3'-thymine moiety of the lesion. On the other hand, yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion, but with markedly reduced efficiency in nucleotide incorporation opposite the 5'-guanine of the lesion. Steady-state kinetic analyses for nucleotide incorporation by yeast pol eta showed that the 5'-guanine portion of the lesion also decreased pronouncedly the fidelity of nucleotide incorporation; the insertion of dAMP and dGMP was favored over that of the correct nucleotide, dCMP. The above results support the conclusion that oxidative intrastrand cross-link lesions, if not repaired, can be cytotoxic and mutagenic.
Jiang
Yong
Y
Environmental Toxicology Graduate Program, University of California at Riverside, Riverside, California 92521, USA.
Hong
Haizheng
H
Cao
Huachuan
H
Wang
Yinsheng
Y
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000528:radiation effects*; D004261:DNA Replication / Q000528:radiation effects*; D005720:Gamma Rays / Q000009:adverse effects*; D006147:Guanine / Q000737:chemistry* / Q000528:radiation effects; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D016296:Mutagenesis / Q000528:radiation effects; D013941:Thymine / Q000737:chemistry* / Q000528:radiation effects
D006147:Guanine; D004247:DNA; D013941:Thymine