title: Efficient and high fidelity incorporation of dCTP opposite 7,8-dihydro-8-oxodeoxyguanosine by Sulfolobus solfataricus DNA polymerase Dpo4.
abstract: DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed >/=95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured K(d)(,dCTP) was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The E(a) for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca(2+). The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5' C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, TN 37232-0146, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Zang
authors.forename: Adriana
authors.initials: A
authors.lastname: Irimia
authors.forename: Jeong-Yun
authors.initials: JY
authors.lastname: Choi
authors.forename: Karen C
authors.initials: KC
authors.lastname: Angel
authors.forename: Lioudmila V
authors.initials: LV
authors.lastname: Loukachevitch
authors.forename: Martin
authors.initials: M
authors.lastname: Egli
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D001483:Base Sequence; D002853:Chromatography, Liquid; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology*; D003845:Deoxycytosine Nucleotides / Q000737:chemistry*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D016368:Frameshift Mutation; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D013997:Time Factors; D014961:X-Ray Diffraction
chemical_list: D017931:DNA Primers; D003845:Deoxycytosine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine