Thermostable DNA polymerase from Thermus thermophilus B35: influence of divalent metal ions on the interaction with deoxynucleoside triphosphates.
The interaction of DNA polymerase from Thermus thermophilus B35 (Tte-pol) with deoxynucleoside triphosphates in the presence of different divalent metal ions has been studied. DNA synthesis and competitive inhibition of the polymerase reaction by non-complementary dNTPs are described with corresponding kinetic schemes. The co-factor properties of some metals (Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Ca2+, Cd2+, and Zn2+) were investigated, and their activating concentration ranges were determined. It was found that kcat values are significantly decreased and Km values slowly decrease when Mn2+ displaces Mg2+. The value of Kd for DNA template-primer is Me2+-independent, whereas Kd values for non-complementary dNTPs decrease in the presence of Mn2+. Tte-pol processivity but not DNA synthesis efficiency is Me2+-type independent.
Rechkunova
N I
NI
Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk. nadyarec@niboch.nsc.ru
Okhapkina
S S
SS
Anarbaev
R O
RO
Lokhova
I A
IA
Degtyarev
S K
SK
Lavrik
O I
OI
D002413:Cations, Divalent; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D016963:Thermus thermophilus / Q000201:enzymology*; D014961:X-Ray Diffraction
D002413:Cations, Divalent; D003854:Deoxyribonucleotides; D004259:DNA-Directed DNA Polymerase