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Conformational dynamics of Thermus aquaticus DNA polymerase I during catalysis.

Abstract：

:Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been performed to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Xu C,Maxwell BA,Suo Z

doi

10.1016/j.jmb.2014.06.003

subject

Has Abstract

pub_date

2014-08-12 00:00:00

pages

2901-2917

issue

16

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(14)00283-6

journal_volume

426

pub_type

杂志文章

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