The one-electron oxidation of guanine in DNA by carbonate radical anions, a decomposition product of peroxynitrosocarbonate which is associated with the inflammatory response, can lead to the formation of intrastrand cross-links between guanine and thymine bases [Crean et al. (Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines. Nucleic Acids Res. 2008; 36: 742-755.)]. These involve covalent bonds between the C8 positions of guanine (G*) and N3 of thymine (T *) in 5′-d(.G *pT *.) and 5′-d(.G *pCpT *.) sequence contexts. We have performed nucleotide excision repair (NER) experiments in human HeLa cell extracts which show that the G *CT * intrastrand cross-link is excised with approximately four times greater efficiency than the G *T * cross-link embedded in 135-mer DNA duplexes. In addition, thermal melting studies reveal that both lesions significantly destabilize duplex DNA, and that the destabilization induced by the G *CT * cross-link is considerably greater. Consistent with this difference in NER, our computations show that both lesions dynamically distort and destabilize duplex DNA. They disturb Watson-Crick base-pairing and base-stacking interactions, and cause untwisting and minor groove opening. These structural perturbations are much more pronounced in the G *CT * than in the G *T * cross-link. Our combined experimental and computational studies provide structural and thermodynamic understanding of the features of the damaged duplexes that produce the most robust NER response.
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