title: Mechanism of the nucleotidyl-transfer reaction in DNA polymerase revealed by time-resolved protein crystallography.
abstract: Nucleotidyl-transfer reaction catalyzed by DNA polymerase is a fundamental enzymatic reaction for DNA synthesis. Until now, a number of structural and kinetic studies on DNA polymerases have proposed a two-metalion mechanism of the nucleotidyl-transfer reaction. However, the actual reaction process has never been visualized. Recently, we have followed the nucleotidyl-transfer reaction process by human DNA polymerase η using time-resolved protein crystallography. In sequence, two Mg(2+) ions bind to the active site, the nucleophile 3'-OH is deprotonated, the deoxyribose at the primer end converts from C2'-endo to C3'-endo, and the nucleophile and the α-phosphate of the substrate dATP approach each other to form the new bond. In this process, we observed transient elements, which are a water molecule to deprotonate the 3'-OH and an additional Mg(2+) ion to stabilize the intermediate state. Particularly, the third Mg(2+) ion observed in this study may be a general feature of the two-metalion mechanism.
authors: Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan; Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors: Teruya
authors: T
authors: Nakamura
authors: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA; Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou 310029, China.
authors: Ye
authors: Y
authors: Zhao
authors: Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.
authors: Yuriko
authors: Y
authors: Yamagata
authors: Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou 310029, China.
authors: Yue-Jin
authors: YJ
authors: Hua
authors: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors: Wei
authors: W
authors: Yang
keywords: DNA polymerase; Mg2+ ion; nucleotidyl-transfer reaction; time-resolved protein crystallography