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dNTP-dependent Conformational Transitions in the Fingers Subdomain of Klentaq1 DNA Polymerase INSIGHTS INTO THE ROLE OF THE "NUCLEOTIDE-BINDING" STATE
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
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2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 19, 13575-13591 p.Article in journal (Refereed) Published
Abstract [en]

DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the "nucleotide-binding" state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state.

Place, publisher, year, edition, pages
2013. Vol. 288, no 19, 13575-13591 p.
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Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-124044DOI: 10.1074/jbc.M112.432690ISI: 000318850300040ScopusID: 2-s2.0-84877707944OAI: diva2:633491

QC 20130627

Available from: 2013-06-27 Created: 2013-06-25 Last updated: 2013-06-27Bibliographically approved

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Sisamakis, EvangelosWidengren, Jerker
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