title: 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.
authors.affiliation: Department of Chemistry, University of Richmond, Gottwald B-100, Richmond, Virginia 23173, United States. mhamm@richmond.edu
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Hamm
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Crowley
authors.forename: Michael
authors.initials: M
authors.lastname: Ghio
authors.forename: Laura
authors.initials: L
authors.lastname: Del Giorno
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Gustafson
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Kindler
authors.forename: Claire W
authors.initials: CW
authors.lastname: Ligon
authors.forename: Maria A M
authors.initials: MA
authors.lastname: Lindell
authors.forename: Emily J
authors.initials: EJ
authors.lastname: McFadden
authors.forename: Carlos
authors.initials: C
authors.lastname: Siekavizza-Robles
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Summers
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D020029:Base Pairing; D004256:DNA Polymerase I / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007700:Kinetics; D009153:Mutagens / Q000737:chemistry / Q000378:metabolism*; D009841:Oligonucleotides / Q000138:chemical synthesis; D013329:Structure-Activity Relationship
chemical_list: D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D009153:Mutagens; D009841:Oligonucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004256:DNA Polymerase I; D003849:Deoxyguanosine; C026600:2'-deoxyadenosine triphosphate