Base selectivity is impaired by mutants that perturb hydrogen bonding networks in the RB69 DNA polymerase active site.
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].
Yang
Guangwei
G
Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, Connecticut 06520, USA.
Wang
Jimin
J
Konigsberg
William
W
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
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