title: Kinetics of mismatch formation opposite lesions by the replicative DNA polymerase from bacteriophage RB69.
abstract: The fidelity of DNA replication is under constant threat from the formation of lesions within the genome. Oxidation of DNA bases leads to the formation of altered DNA bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxoG, and 2-hydroxyadenine, or 2-OHA. In this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analogue by the replicative DNA polymerase from bacteriophage RB69. We compared the kinetic parameters for both wild type and the low fidelity L561A variant. While nucleotide incorporation rates (k(pol)) were generally higher for the variant, the presence of a lesion in the templating position reduced the ability of both the wild-type and variant DNA polymerases to form ternary enzyme-DNA-dNTP complexes. Thus, the L561A substitution does not significantly affect the ability of the RB69 DNA polymerase to recognize damaged DNA; instead, the mutation increases the probability that nucleotide incorporation will occur. We have also solved the crystal structure of the L561A variant forming an 8-oxoG.dATP mispair and show that the propensity for forming this mispair depends on an enlarged polymerase active site.
authors.affiliation: Department of Microbiology and Molecular Genetics, 95 Carrigan Drive, University of Vermont, Burlington, Vermont 05405, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Hogg
authors.forename: Jean
authors.initials: J
authors.lastname: Rudnicki
authors.forename: John
authors.initials: J
authors.lastname: Midkiff
authors.forename: Linda
authors.initials: L
authors.lastname: Reha-Krantz
authors.forename: Sylvie
authors.initials: S
authors.lastname: Doublié
authors.forename: Susan S
authors.initials: SS
authors.lastname: Wallace
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001435:Bacteriophages / Q000201:enzymology*; D020137:Base Pair Mismatch; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase