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An intramolecular FRET system monitors fingers subdomain opening in Klentaq1

Allen, William J. and Rothwell, P.J. and Waksman, Gabriel (2008) An intramolecular FRET system monitors fingers subdomain opening in Klentaq1. Protein Science 17 (3), pp. 401-408. ISSN 0961-8368.

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Official URL: http://dx.doi.org/10.1110/ps.073309208

Abstract

A major goal of polymerase research is to determine the mechanism through which a nucleotide complementary to a templating DNA base is selected and delivered to the polymerase active site. Structural evidence suggests a large open-to-closed conformational change affecting the fingers subdomain as being crucial to the process. We previously designed a FRET system capable of measuring the rate of fingers subdomain closure in the presence of correct nucleotide. However, this FRET system was limited in that it could not directly measure the rate of fingers subdomain opening by FRET after polymerization or in the absence of DNA. Here we report the development of a new system capable of measuring both fingers subdomain closure and reopening by FRET, and show that the rate of fingers subdomain opening is limited only by the rate of polymerization. We anticipate that this system will scale down to the single molecule level, allowing measurement of fingers subdomain movements in the presence of incorrect nucleotide and in the absence of DNA.

Item Type: Article
Keyword(s) / Subject(s): polymerase, Klentaq1, subdomain movement, FRET, stopped-flow, kinetics
School or Research Centre: Birkbeck Schools and Research Centres > School of Science > Biological Sciences
Depositing User: Administrator
Date Deposited: 04 Aug 2010 14:09
Last Modified: 17 Apr 2013 12:17
URI: http://eprints.bbk.ac.uk/id/eprint/1156

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