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2-aminopurine fluorescence, wild-type, replication fidelity, nucleotide incorporation, biochemical basis, deoxyribonucleic acid polymerases, template strand, primer extension, exonuclease activity, spontaneous mutation

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Subject-Keyword: 2-aminopurine fluorescence wild-type replication fidelity nucleotide incorporation biochemical basis deoxyribonucleic acid polymerases template strand primer extension exonuclease activity spontaneous mutation

Type of item: Journal Article Published

Language: English

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Description: 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 polymeraseto- 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 s21; ii T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonucleaseto- polymerase and polymerase-to-exonuclease active site switching steps and iii proofreading reactions that initiate in the polymerase active center are not intrinsically processive.

Date created: 2007

DOI: doi:10.7939-R30C4SN3N

License information: This item is open access. The source

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Rights: This item is open access. The source and authors must be cited.





Autor: da Silva, E.F. Reha-Krantz, L.J.

Fuente: https://era.library.ualberta.ca/


Introducción



5452–5463 Nucleic Acids Research, 2007, Vol.
35, No.
16 doi:10.1093-nar-gkm591 Published online 15 August 2007 DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase Elizabeth Fidalgo da Silva and Linda J.
Reha-Krantz* Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada Received April 17, 2007; Revised June 9, 2007; Accepted July 18, 2007 ABSTRACT INTRODUCTION DNA polymerase proofreading removes misincorporated nucleotides at the primer-end (1,2), which significantly improves the fidelity of DNA replication (3).
Since increased epithelial tumors are observed in mice that express an exonuclease-deficient DNA polymerase d, DNA polymerase proofreading is important in preventing mutations that lead to cancer (4).
DNA polymerase proofreading was first demonstrated to be a major determinant of replication fidelity for the bacteriophage T4 DNA polymerase (2,5,6) and this DNA polymerase *To whom correspondence should be addressed.
Tel: 1 780 492 5383; Fax: 1 780 492 9234; Email: Linda.Reha-Krantz@ualberta.ca ß 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. Downloaded from http:--nar.oxfordjournals.org- at University Of Alberta Library on March 9, 2012 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 ...





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