Effect of Different Divalent Cations on the Kinetics and Fidelity of RB69 DNA Polymerase.
Although Mg(2+) is the cation that functions as the cofactor for the nucleotidyl transfer reaction for almost all DNA polymerases, Mn(2+) can also serve, but when it does, the degree of base discrimination exhibited by most DNA polymerases (pols) is diminished. Metal ions other than Mg(2+) or Mn(2+) can also act as cofactors depending on the specific DNA polymerase. Here, we tested the ability of several divalent metal ions to substitute for Mg(2+) or Mn(2+) with RB69 DNA polymerase (RB69pol), a model B-family pol. Our choice of metal ions was based on previous studies with other DNA pols. Co(2+), and to a lesser extent Ni(2+), were the only cations among those tested besides Mg(2+) and Mn(2+) that could serve as cofactors with RB69pol. The incorporation efficiency of correct dNMPs increased by 5-fold with Co(2+), relative to that of Mg(2+). The incorporation efficiencies of incorrect dNMPs increased by 2-17-fold with Co(2+), relative to that with Mg(2+) depending on the incoming dNTP. Base selectivity was reduced even further with Mn(2+) compared to that observed with Co(2+). Substitution of Mn(2+), Co(2+), or Ni(2+) for Mg(2+) reduced the exonuclease activity of RB69pol by 2-, 6-, and 33-fold, respectively, contributing to the frequency of misincorporation. In addition, Co(2+) and Mn(2+) were better able to extend a primer past a mismatch than Mg(2+). Finally, Co(2+) and Mn(2+) enhanced ground-state binding of both correct and incorrect dNTPs to RB69pol:dideoxy-terminated primer-template complexes.
Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06520-8024, United States.
Ashwani Kumar
AK
Vashishtha
Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06520-8024, United States.
William H
WH
Konigsberg
D001435:Bacteriophages / Q000201:enzymology*; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000821:virology; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D002413:Cations, Divalent; D009711:Nucleotides; D014764:Viral Proteins; D003035:Cobalt; D008345:Manganese; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase; D008274:Magnesium