Mechanism of the nucleotidyl-transfer reaction in DNA polymerase revealed by time-resolved protein crystallography

• Nakamura Teruya

  Graduate School of Pharmaceutical Sciences, Kumamoto University Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health

• Zhao Ye

  Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Institute of Nuclear-Agricultural Sciences, Zhejiang University

• Yamagata Yuriko

  Graduate School of Pharmaceutical Sciences, Kumamoto University

• Hua Yue-jin

  Institute of Nuclear-Agricultural Sciences, Zhejiang University

• Yang Wei

  Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health

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-metal-ion 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 Mg²⁺ 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 Mg²⁺ ion to stabilize the intermediate state. Particularly, the third Mg²⁺ ion observed in this study may be a general feature of the two-metal-ion mechanism.<br>

Journal

• BIOPHYSICS

  BIOPHYSICS 9 (0), 31-36, 2013

  The Biophysical Society of Japan

Keywords

• DNA polymerase
• nucleotidyl-transfer reaction
• Mg²⁺ ion
• time-resolved protein crystallography

Details

• CRID: 1390282680198819328
• NII Article ID: 130003366027
• DOI: 10.2142/biophysics.9.31
• ISSN: 13492942
• Web Site: https://www.jstage.jst.go.jp/article/biophysics/9/0/9_31/_pdf
• Text Lang: en
• Data Source:
  • JaLC
  • Crossref
  • CiNii Articles
  • KAKEN
• Abstract License Flag: Disallowed