Differences in replication of a DNA template containing an ethyl phosphotriester by T4 DNA polymerase and Escherichia coli DNA polymerase IReport as inadecuate




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deoxyribonucleic acid polymerases, exonuclease activity, antimutator, 2-aminopurine fluorescence, sites, wild-type, repair, bacteriophage-T4, nucleotide incorporation, methyl phosphotriesters

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Subject-Keyword: deoxyribonucleic acid polymerases exonuclease activity antimutator 2-aminopurine fluorescence sites wild-type repair bacteriophage-T4 nucleotide incorporation methyl phosphotriesters

Type of item: Journal Article Published

Language: English

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Description: A DNA template containing a single ethyl phosphotriester was replicated in vitro by the bacteriophage T4 DNA polymerase and by Escherichia coli DNA polymerase I DNA pol I. Escherichia coli DNA pol I bypassed the lesion ef®ciently, but partial inhibition was observed for T4 DNA polymerase. The replication block produced by the ethyl phosphotriester was increased at low dNTP concentrations and for a mutant T4 DNA polymerase with an antimutator phenotype, increased proofreading activity, and reduced ability to bind DNA in the polymerase active center. These observations support a model in which an ethyl phosphotriester impedes primer elongation by T4 DNA polymerase by decreasing formation of the ternary DNA polymerase±DNA± dNTP complex. When primer elongation is not possible, proofreading becomes the favored reaction. Apparent futile cycles of nucleotide incorporation and proofreading, the idling reaction, were observed at the site of the lesion. The replication block was overcome by higher dNTP concentrations. Thus, ethyl phosphotriesters may be tolerated in vivo by the up-regulation of dNTP biosynthesis that occurs during the cellular checkpoint response to blocked DNA replication forks.

Date created: 2003

DOI: doi:10.7939-R3445HF69

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Rights: © Oxford University Press 2003; all rights reserved





Author: Tsujikawa, L. Weinfield, M. Reha-Krantz, L.J.

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


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Nucleic Acids Research, 2003, Vol.
31, No.
17 4965±4972 DOI: 10.1093-nar-gkg722 Differences in replication of a DNA template containing an ethyl phosphotriester by T4 DNA polymerase and Escherichia coli DNA polymerase I Laura Tsujikawa, Michael Wein®eld1 and Linda J.
Reha-Krantz* Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada and 1 Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada Received June 12, 2003; Revised and Accepted July 17, 2003 A DNA template containing a single ethyl phosphotriester was replicated in vitro by the bacteriophage T4 DNA polymerase and by Escherichia coli DNA polymerase I (DNA pol I).
Escherichia coli DNA pol I bypassed the lesion ef®ciently, but partial inhibition was observed for T4 DNA polymerase.
The replication block produced by the ethyl phosphotriester was increased at low dNTP concentrations and for a mutant T4 DNA polymerase with an antimutator phenotype, increased proofreading activity, and reduced ability to bind DNA in the polymerase active center.
These observations support a model in which an ethyl phosphotriester impedes primer elongation by T4 DNA polymerase by decreasing formation of the ternary DNA polymerase±DNA± dNTP complex.
When primer elongation is not possible, proofreading becomes the favored reaction. Apparent futile cycles of nucleotide incorporation and proofreading, the idling reaction, were observed at the site of the lesion.
The replication block was overcome by higher dNTP concentrations.
Thus, ethyl phosphotriesters may be tolerated in vivo by the up-regulation of dNTP biosynthesis that occurs during the cellular checkpoint response to blocked DNA replication forks. INTRODUCTION A variety of adducts are produced in DNA by exposure to endogenous and exogenous alkylating agents.
Some of the most abundant lesions formed are 7-alkylguanine, O6alkylguanine, 3-alkyladenine and alkyl phosphotriesters. While 7-methylguanine and 3-met...





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