Saturno J, Blanco L, Salas M, Esteban J A
Centro de Biología Molecular, Severo Ochoa (Consejo Superior de Investigaciones Ceintíficas-Universidad Autonoma de Madrid), Spain.
J Biol Chem. 1995 Dec 29;270(52):31235-43. doi: 10.1074/jbc.270.52.31235.
Amino acids Tyr254 and Tyr390 of phi 29 DNA polymerase belong to one of the most conserved regions in eukaryotic-type DNA polymerases. In this paper we report a mutational study of these two residues to address their role in nucleotide selection. This study was carried out by means of a new kinetic analysis that takes advantage of the competition between DNA polymerization and 3'-->5' exonuclease activity to measure the Km values for correct and incorrect nucleotides in steady-state conditions. This method is valid for any 3'-->5' exonuclease-containing DNA polymerase, without any restriction concerning catalytic rates of nucleotide incorporation. The results showed that the discrimination factor achieved by phi 29 DNA polymerase in the nucleotide binding step of DNA polymerization is 2.4 x 10(3), that is, a wrong nucleotide is bound with a 2.4 x 10(3)-fold lower affinity than the correct one. Mutants Y254F, Y390F, and Y390S showed discrimination values of 7.0 x 10(2), > 1.9 x 10(3), and 2.9 x 10(2), respectively. The reduced accuracy of nucleotide binding produced by mutations Y254F and Y390S lead us to propose that phi 29 DNA polymerase residues Tyr254 and Tyr390, highly conserved in eukaryotic-type DNA polymerases, are involved in nucleotide binding selection, thus playing a crucial role in the fidelity of DNA replication. Comparison of the discrimination factors of mutants Y390S and Y390F strongly suggests that the phenyl ring of Tyr390 is directly involved in checking base-pairing correctness of the incoming nucleotide.
phi 29 DNA聚合酶的酪氨酸254和酪氨酸390属于真核生物型DNA聚合酶中最保守的区域之一。在本文中，我们报告了对这两个残基的突变研究，以探讨它们在核苷酸选择中的作用。这项研究是通过一种新的动力学分析方法进行的，该方法利用DNA聚合和3'→5'核酸外切酶活性之间的竞争来测量稳态条件下正确和错误核苷酸的Km值。这种方法对任何含有3'→5'核酸外切酶的DNA聚合酶都是有效的，不受核苷酸掺入催化速率的任何限制。结果表明，phi 29 DNA聚合酶在DNA聚合的核苷酸结合步骤中实现的区分因子为2.4×10³，即错误核苷酸的结合亲和力比正确核苷酸低2.4×10³倍。突变体Y254F、Y390F和Y390S的区分值分别为7.0×10²、>1.9×10³和2.9×10²。Y254F和Y390S突变导致的核苷酸结合准确性降低使我们提出，在真核生物型DNA聚合酶中高度保守的phi 29 DNA聚合酶残基酪氨酸254和酪氨酸390参与核苷酸结合选择，从而在DNA复制的保真度中起关键作用。突变体Y390S和Y390F区分因子的比较强烈表明，酪氨酸390的苯环直接参与检查进入核苷酸的碱基配对正确性。
