title: Base selectivity is impaired by mutants that perturb hydrogen bonding networks in the RB69 DNA polymerase active site.
abstract: To investigate the molecular basis for the selective utilization of nucleoside triphosphates complementary to templating bases, by RB69 DNA polymerase (RB69 pol), we constructed a set of mutants that we predicted would perturb the "floor" of the nascent base-pairing interface in the enzyme. We then determined the pre-steady-state kinetic parameters for the incorporation of complementary and noncomplementary dNTPs by the exo(-) form of RB69 pol and its mutants. We found that the Y567A mutant had the same K(d) and k(pol) values for incorporation of C versus G as the wild-type exo(-) enzyme; however, the k(pol)/K(d) ratio for G versus G incorporation with the Y567A mutant was 10 times higher than the k(pol)/K(d) efficiency of G versus G incorporation using the exo(-) RB69 pol. The reduced level of discrimination by the Y567A mutant against incorporation of mismatched bases was also seen with the Y391A mutant. Stopped-flow fluorescence was also employed to monitor rates of putative conformational changes with the exo(-) RB69 pol and its mutants using a primer-template complex containing 2-aminopurine. The rates of fluorescence changes were equal to or greater than the rates of the rapid chemical quench, indicating that we were monitoring a process occurring before or during the phosphoryl transfer reaction. We have interpreted our results within the context of the crystal structure of the RB69 pol ternary complex [Franklin, M. C., et al. (2001) Cell 105, 657-667].
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, Connecticut 06520, USA.
authors.forename: Guangwei
authors.initials: G
authors.lastname: Yang
authors.forename: Jimin
authors.initials: J
authors.lastname: Wang
authors.forename: William
authors.initials: W
authors.lastname: Konigsberg
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004754:Enterobacter / Q000235:genetics* / Q000821:virology*; D006860:Hydrogen Bonding; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013942:Thymine Nucleotides / Q000378:metabolism; D014050:Toluene / Q000031:analogs & derivatives* / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: C110637:2'-deoxy-1'-(2,4-difluorotol-5-yl)ribofuranose 5'-triphosphate; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D009711:Nucleotides; D013942:Thymine Nucleotides; D014764:Viral Proteins; C024107:2'-deoxycytidine 5'-triphosphate; D014050:Toluene; D014443:Tyrosine; D010649:Phenylalanine; C029603:deoxyguanosine triphosphate; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase; C026600:2'-deoxyadenosine triphosphate; D000409:Alanine; C024157:thymidine 5'-triphosphate