Importance of the C2, N7, and C8 Positions to the Mutagenic Potential of 8-Oxo-2′-deoxyguanosine with Two A Family Polymerases

Abstract

8-Oxo-2'-deoxyguanosine (OdG) is a prominent DNA lesion produced from the reaction of 2'-deoxyguanosine (dG) with reactive oxygen species. While dG directs the insertion of only dCTP during replication, OdG can direct the insertion of either dCTP or dATP, allowing for the production of dG → dT transversions. When replicated by Klenow fragment-exo (KF-exo), OdG preferentially directs the incorporation of dCTP over dATP, thus decreasing its mutagenic potential. However, when replicated by a highly related polymerase, the large fragment of polymerase I from Bacillus stearothermophilus (BF), dATP incorporation is preferred, and a higher mutagenic potential results. To gain insight into the reasons for this opposite preference and the effects of the C2, N7, and C8 positions on OdG mutagenicity, single-nucleotide insertions of dCTP and/or dATP opposite dG, OdG, and seven of their analogues were examined by steady state kinetics with both KF-exo and BF. Results from these studies suggest that the two enzymes behave similarly and are both sensitive not only to steric and electronic changes within the imidazole ring during both dCTP and dATP incorporation but also to the presence of the C2-exocyclic amine during dATP incorporation. The difference in incorporation preference opposite OdG appears to be due to a somewhat increased sensitivity to structural perturbations during dCTP incorporation with BF. Single-nucleotide extensions past the resulting base pairs were also studied and were not only similar between the two enzymes but also consistent with published ternary crystallographic studies with BF. These results are analyzed in the context of previous biochemical and structural studies, as well as stability studies with the resulting base pairs.

Structure–Activity Relationships Reveal Key Features of 8-Oxoguanine: A Mismatch Detection by the MutY Glycosylase

“…29 However, 9ZG:A pairs are more stable to thermal denaturation than G:A mismatches because 9ZG retains a hydrogen bond donor at N7 that can help stabilize the Hoogsteen pairing to A. 30 …”

“…8-Thioguanine (8SG) displays an increase in the bulk of the 8-substituent along with a mild decrease in that substituent’s electronegativity; this analog shows similar conformational and base pairing proclivities to OG. 30 The base of 8-oxoinosine (8OI) lacks the exocyclic amine of OG, producing a Watson–Crick face that typically codes like G under experimental conditions. 31 Likewise, duplexes containing 8SG:A and 8OI:A pairs have similar duplex stabilities to those harboring OG:A pairs.…”

“…31 Likewise, duplexes containing 8SG:A and 8OI:A pairs have similar duplex stabilities to those harboring OG:A pairs. 30 …”

Polymerase bypass of N7-guanine monoadducts of cisplatin, diepoxybutane, and epichlorohydrin

“…The single-stranded template was then annealed to a primer, and DNA synthesis studies were performed with three different polymerases: Klenow fragment from Escherichia coli (KF), the large fragment of DNA Polymerase I from Bacillus stearothermophilus (BF), and recombinant human polymerase β (hPol β). The former are two related A-family bacterial polymerases commonly used as model enzymes in lesion bypass studies [24, 25], and the latter is a replicative X-family polymerase. Primers terminated two nucleotides before the lesion, one nucleotide before the lesion, or at the lesion in order to assess whether any effect of the lesion is highly local or also affects synthesis at flanking bases.…”

Insights into the substrate specificity of the MutT pyrophosphohydrolase using structural analogues of 8-oxo-2′-deoxyguanosine nucleotide

“…Though we have previously incorporated 8-thio-2′-deoxyinosine (SdI) into oligonucleotides using phosphoramidite chemistry, this study necessitated the mononucleoside, which we had not previously synthesized. Therefore, we first obtained SdI monomer from our previously characterized 8-thio protected SdI monomer 23 through deprotection using tetrabutylammonium fluoride (TBAF); we then synthesized SdITP using salicyl chlorophosphite (2-chloro-1,3,2-benzodioxaphosphorin-4-one) and tributylammonium pyrophosphate (Fig. 3B).…”