Thermostable DNA Polymerase from Thermus thermophilus B35: Influence of Divalent Metal Ions on the Interaction with Deoxynucleoside Triphosphates

N. I. Rechkunova^1*, S. S. Okhapkina², R. O. Anarbaev¹, I. A. Lokhova¹, S. Kh. Degtyarev³, and O. I. Lavrik¹

¹Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, pr. Lavrent'eva 8, Novosibirsk, 630090 Russia; fax: (3832) 33-3677; E-mail: nadyarec@niboch.nsc.ru

²Novosibirsk State University, ul. Pirogova 2, Novosibirsk, 630090 Russia

³Institute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Medical Sciences, ul. Timakova 2, Novosibirsk, 630117 Russia; fax: (3832) 33-6853

^* To whom correspondence should be addressed.

Received July 15, 1999; Revision received January 31, 2000

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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 k_cat values are significantly decreased and K_m values slowly decrease when Mn²⁺ displaces Mg²⁺. The value of K_d for DNA template-primer is Me²⁺-independent, whereas K_d values for non-complementary dNTPs decrease in the presence of Mn²⁺. Tte-pol processivity but not DNA synthesis efficiency is Me²⁺-type independent.

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KEY WORDS: thermostable DNA polymerase, deoxynucleoside triphosphates, divalent metal ions, kinetic parameters

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