Assessment of the genotoxic potential of nitric oxide-induced guanine lesions by in vitro reactions with Escherichia coli DNA polymerase I.

Abstract:

It has been suggested that carcinogenesis associated with chronic inflammation involves DNA damage by nitric oxide (NO) and other reactive species secreted from macrophages and neutrophils. The guanine moiety of DNA reacts with NO, yielding two major deamination products: xanthine (Xan) and oxanine (Oxa). Oxa reacts further with polyamines and DNA binding proteins to form cross-link adducts. In the present study, we characterized the structure of the cross-link adducts of Oxa with spermine (Oxa-Sp). Spectrometric analysis of Oxa-Sp adducts showed that they are ring-opened adducts of Oxa covalently bonded to the terminal amino (major product) and internal imino (minor product) groups of spermine. To assess genotoxic potential, Xan, Oxa, Oxa-Sp and an abasic (AP) site were site specifically incorporated into oligonucleotide templates. These lesions differentially blocked in vitro DNA synthesis catalyzed by DNA polymerase I Klenow fragment (Pol I Kf). The relative efficiency of translesion synthesis was G (1) > Oxa (0.19) > Xan (0.12) > AP (0.088) > Oxa-Sp (0.035). Primer extension assays with a single nucleotide and Pol I Kf revealed that non-mutagenic dCMP was inserted most efficiently opposite Xan and Oxa, with the extent of primer elongation being 65% for Xan and 68% for Oxa. However, mutagenic nucleotides were also inserted. The extent of primer elongation for Xan was 16% with dTMP and 14% with dGMP, whereas that for Oxa was 49% with dTMP. For Oxa-Sp, mutagenic dAMP (13%) was preferentially inserted. Accordingly, when generated in vivo, Xan and Oxa would constitute moderate blocks to DNA synthesis and primarily elicit G:C to A:T transitions when bypassed, whereas Oxa-Sp would strongly block DNA synthesis and elicit G:C to T:A transversions.

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