The function of a lysine residue, Lys, of human DNA polymerase located in the third most conserved region and conserved in all of the -like polymerases was analyzed by site-directed mutagenesis. Lys was mutagenized to Arg, Ala, or Asn. The mutant enzymes were expressed in insect cells infected with recombinant baculoviruses and purified to near homogeneity. The mutant enzymes had specific activities ranging from 8 to 22% of the wild type. All three Lys mutants utilized Mn as metal activator more effectively than the wild type enzyme and showed an increase in Kvalues for deoxynucleoside triphosphate but not k values in reactions with either Mg or Mn as the metal activator. Although mutation of the Lys residue caused an increase in K values for deoxynucleoside triphosphates, mutations of Lys to Arg, Ala, or Asn did not alter the mutant enzymes' misinsertion efficiency in reactions with Mg as a metal activator as compared with that of the wild type, suggesting that the base of the incoming deoxynucleoside triphosphate is not the structural feature interacting with the Lys side chain. In reaction with Mn as a metal activator, all three Lys mutants had an improved fidelity for deoxynucleotide misinsertion compared to wild type. Inhibition studies of the three Lys mutant derivatives with an inhibitor, structural analogs of deoxynucleoside triphosphate, and pyrophosphate suggest that the deoxyribose sugar and -,-phosphate groups are not the structural feature recognized by the Lys side chain. Comparison of the mutant enzymes to the wild type enzyme for their affinities for dCTPS versus deoxynucleoside triphosphate suggests that this highly conserved Lys is involved in interacting either directly or indirectly with the oxygen moiety of the -phosphate of the incoming deoxynucleoside triphosphate.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				C. A. Howell, C. M. Kondratick, and M. T. Washington Substitution of a residue contacting the triphosphate moiety of the incoming nucleotide increases the fidelity of yeast DNA polymerase {zeta} Nucleic Acids Res., March 1, 2008; 36(5): 1731 - 1740. [Abstract] [Full Text] [PDF]

				Y. T. Hwang, H. J. Zuccola, Q. Lu, and C. B. C. Hwang A Point Mutation within Conserved Region VI of Herpes Simplex Virus Type 1 DNA Polymerase Confers Altered Drug Sensitivity and Enhances Replication Fidelity J. Virol., January 15, 2004; 78(2): 650 - 657. [Abstract] [Full Text] [PDF]

				P. J. A. Gutierrez and T. S.-F. Wang Genomic Instability Induced by Mutations in Saccharomyces cerevisiae POL1 Genetics, September 1, 2003; 165(1): 65 - 81. [Abstract] [Full Text] [PDF]

				M. Ogawa, S. Limsirichaikul, A. Niimi, S. Iwai, S. Yoshida, and M. Suzuki Distinct Function of Conserved Amino Acids in the Fingers of Saccharomyces cerevisiae DNA Polymerase {alpha} J. Biol. Chem., May 23, 2003; 278(21): 19071 - 19078. [Abstract] [Full Text] [PDF]

				S. Limsirichaikul, M. Ogawa, A. Niimi, S. Iwai, T. Murate, S. Yoshida, and M. Suzuki The Gly-952 Residue of Saccharomyces cerevisiae DNA Polymerase {alpha} Is Important in Discriminating Correct Deoxyribonucleotides from Incorrect Ones J. Biol. Chem., May 23, 2003; 278(21): 19079 - 19086. [Abstract] [Full Text] [PDF]

				H. Liu, J. H. Naismith, and R. T. Hay Identification of Conserved Residues Contributing to the Activities of Adenovirus DNA Polymerase J. Virol., December 15, 2000; 74(24): 11681 - 11689. [Abstract] [Full Text]

				S. J. Evans, M. J. Fogg, A. Mamone, M. Davis, L. H. Pearl, and B. A. Connolly Improving dideoxynucleotide-triphosphate utilisation by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus Nucleic Acids Res., March 1, 2000; 28(5): 1059 - 1066. [Abstract] [Full Text] [PDF]

				L. Huang, K. K. Ishii, H. Zuccola, A. M. Gehring, C. B. C. Hwang, J. Hogle, and D. M. Coen The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: Relationship to the structure of alpha -like DNA polymerases PNAS, January 19, 1999; 96(2): 447 - 452. [Abstract] [Full Text] [PDF]

				S.-M. Wu, P. Zhang, X. R. Zeng, S.-J. Zhang, J. Mo, B. Q. Li, and M. Y. W. T. Lee Characterization of the p125 Subunit of Human DNA Polymerase delta  and Its Deletion Mutants. INTERACTION WITH CYCLIN-DEPENDENT KINASE-CYCLINS J. Biol. Chem., April 17, 1998; 273(16): 9561 - 9569. [Abstract] [Full Text] [PDF]

				A. Monaghan and R. T. Hay Pyridoxal 5'-Phosphate Inhibition of Adenovirus DNA Polymerase J. Biol. Chem., September 27, 1996; 271(39): 24242 - 24248. [Abstract] [Full Text] [PDF]