title: The anti/syn conformation of 8-oxo-7,8-dihydro-2'-deoxyguanosine is modulated by Bacillus subtilis PolX active site residues His255 and Asn263. Efficient processing of damaged 3'-ends.
abstract: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG) is a major lesion resulting from oxidative stress and found in both DNA and dNTP pools. Such a lesion is usually removed from DNA by the Base Excision Repair (BER), a universally conserved DNA repair pathway. 8oxodG usually adopts the favored and promutagenic syn-conformation at the active site of DNA polymerases, allowing the base to hydrogen bonding with adenine during DNA synthesis. Here, we study the structural determinants that affect the glycosidic torsion-angle of 8oxodGTP at the catalytic active site of the family X DNA polymerase from Bacillus subtilis (PolXBs). We show that, unlike most DNA polymerases, PolXBs exhibits a similar efficiency to stabilize the anti and syn conformation of 8oxodGTP at the catalytic site. Kinetic analyses indicate that at least two conserved residues of the nucleotide binding pocket play opposite roles in the anti/syn conformation selectivity, Asn263 and His255 that favor incorporation of 8oxodGMP opposite dA and dC, respectively. In addition, the presence in PolXBs of Mn2+-dependent 3'-phosphatase and 3'-phosphodiesterase activities is also shown. Those activities rely on the catalytic center of the C-terminal Polymerase and Histidinol Phosphatase (PHP) domain of PolXBs and, together with its 3'-5' exonuclease activity allows the enzyme to resume gap-filling after processing of damaged 3' termini.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Nicolás Cabrera 1, 28049 Madrid, Spain.
authors.forename: Olga
authors.initials: O
authors.lastname: Zafra
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Nicolás Cabrera 1, 28049 Madrid, Spain.
authors.forename: Lucía
authors.initials: L
authors.lastname: Pérez de Ayala
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Nicolás Cabrera 1, 28049 Madrid, Spain. Electronic address: mdevega@cbm.csic.es.
authors.forename: Miguel
authors.initials: M
authors.lastname: de Vega
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D016415:Sequence Alignment
chemical_list: D004269:DNA, Bacterial; D000080242:8-Hydroxy-2'-Deoxyguanosine; C109534:DNA polymerase X; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine
keywords: 8-Oxoguanine; DNA repair; Enzyme kinetics; Family X DNA polymerase; Phosphatase; Phosphodiesterase