In vivo formation and in vitro replication of a guanine-thymine intrastrand cross-link lesion

Biochemistry. 2007 Nov 6;46(44):12757-63. doi: 10.1021/bi7012195. Epub 2007 Oct 12.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Base Sequence
  • DNA / chemistry
  • DNA / radiation effects*
  • DNA Replication / radiation effects*
  • Gamma Rays / adverse effects*
  • Guanine / chemistry*
  • Guanine / radiation effects
  • HeLa Cells
  • Humans
  • Models, Biological
  • Mutagenesis* / radiation effects
  • Thymine / chemistry*
  • Thymine / radiation effects

Substances

  • Guanine
  • DNA
  • Thymine