Nucleic Acids Research Advance Access originally published online on August 15, 2007
Nucleic Acids Research 2007 35(16):5452-5463; doi:10.1093/nar/gkm591
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Nucleic Acids Research, 2007, Vol. 35, No. 16 5452-5463
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
*To whom correspondence should be addressed. Tel: +1 780 492 5383; Fax: +1 780 492 9234; Email: Linda.Reha-Krantz{at}ualberta.ca
Received April 17, 2007. Revised June 9, 2007. Accepted July 18, 2007.
DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center. Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine (2AP) to measure the rates of active site switching for the bacteriophage T4 DNA polymerase. There are three findings: (i) the rate of return of the trimmed primer-end from the exonuclease to the polymerase active center is rapid, >500 s–1; (ii) T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonuclease-to-polymerase and polymerase-to-exonuclease active site switching steps and (iii) proofreading reactions that initiate in the polymerase active center are not intrinsically processive.