In Vivo Formation and in Vitro Replication of a Guanine−Thymine Intrastrand Cross-Link Lesion†
datasetposted on 06.11.2007 by Yong Jiang, Haizheng Hong, Huachuan Cao, Yinsheng Wang
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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 γ- 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 γ-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 η (pol η) 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 η 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.