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Prechemistry Nucleotide Selection Checkpoints in the Reaction Pathway of DNA Polymerase I and Roles of Glu710 and Tyr766
[Image: see text] The accuracy of high-fidelity DNA polymerases such as DNA polymerase I (Klenow fragment) is governed by conformational changes early in the reaction pathway that serve as fidelity checkpoints, identifying inappropriate template–nucleotide pairings. The fingers-closing transition (d...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2013
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3770053/ https://www.ncbi.nlm.nih.gov/pubmed/23937394 http://dx.doi.org/10.1021/bi400837k |
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author | Bermek, Oya Grindley, Nigel D. F. Joyce, Catherine M. |
author_facet | Bermek, Oya Grindley, Nigel D. F. Joyce, Catherine M. |
author_sort | Bermek, Oya |
collection | PubMed |
description | [Image: see text] The accuracy of high-fidelity DNA polymerases such as DNA polymerase I (Klenow fragment) is governed by conformational changes early in the reaction pathway that serve as fidelity checkpoints, identifying inappropriate template–nucleotide pairings. The fingers-closing transition (detected by a fluorescence resonance energy transfer-based assay) is the unique outcome of binding a correct incoming nucleotide, both complementary to the templating base and with a deoxyribose (rather than ribose) sugar structure. Complexes with mispaired dNTPs or complementary rNTPs are arrested at an earlier stage, corresponding to a partially closed fingers conformation, in which weak binding of DNA and nucleotide promote dissociation and resampling of the substrate pool. A 2-aminopurine fluorescence probe on the DNA template provides further information about the steps preceding fingers closing. A characteristic 2-aminopurine signal is observed on binding a complementary nucleotide, regardless of whether the sugar is deoxyribose or ribose. However, mispaired dNTPs show entirely different behavior. Thus, a fidelity checkpoint ahead of fingers closing is responsible for distinguishing complementary from noncomplementary nucleotides and routing them toward different outcomes. The E710A mutator polymerase has a defect in the early fidelity checkpoint such that some complementary dNTPs are treated as if they were mispaired. In the Y766A mutant, the early checkpoint functions normally, but some correctly paired dNTPs do not efficiently undergo fingers closing. Thus, both mutator alleles cause a blurring of the distinction between correct and incorrect base pairs and result in a larger fraction of errors passing through the prechemistry fidelity checkpoints. |
format | Online Article Text |
id | pubmed-3770053 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-37700532013-09-13 Prechemistry Nucleotide Selection Checkpoints in the Reaction Pathway of DNA Polymerase I and Roles of Glu710 and Tyr766 Bermek, Oya Grindley, Nigel D. F. Joyce, Catherine M. Biochemistry [Image: see text] The accuracy of high-fidelity DNA polymerases such as DNA polymerase I (Klenow fragment) is governed by conformational changes early in the reaction pathway that serve as fidelity checkpoints, identifying inappropriate template–nucleotide pairings. The fingers-closing transition (detected by a fluorescence resonance energy transfer-based assay) is the unique outcome of binding a correct incoming nucleotide, both complementary to the templating base and with a deoxyribose (rather than ribose) sugar structure. Complexes with mispaired dNTPs or complementary rNTPs are arrested at an earlier stage, corresponding to a partially closed fingers conformation, in which weak binding of DNA and nucleotide promote dissociation and resampling of the substrate pool. A 2-aminopurine fluorescence probe on the DNA template provides further information about the steps preceding fingers closing. A characteristic 2-aminopurine signal is observed on binding a complementary nucleotide, regardless of whether the sugar is deoxyribose or ribose. However, mispaired dNTPs show entirely different behavior. Thus, a fidelity checkpoint ahead of fingers closing is responsible for distinguishing complementary from noncomplementary nucleotides and routing them toward different outcomes. The E710A mutator polymerase has a defect in the early fidelity checkpoint such that some complementary dNTPs are treated as if they were mispaired. In the Y766A mutant, the early checkpoint functions normally, but some correctly paired dNTPs do not efficiently undergo fingers closing. Thus, both mutator alleles cause a blurring of the distinction between correct and incorrect base pairs and result in a larger fraction of errors passing through the prechemistry fidelity checkpoints. American Chemical Society 2013-08-12 2013-09-10 /pmc/articles/PMC3770053/ /pubmed/23937394 http://dx.doi.org/10.1021/bi400837k Text en Copyright © 2013 American Chemical Society Terms of Use (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) |
spellingShingle | Bermek, Oya Grindley, Nigel D. F. Joyce, Catherine M. Prechemistry Nucleotide Selection Checkpoints in the Reaction Pathway of DNA Polymerase I and Roles of Glu710 and Tyr766 |
title | Prechemistry Nucleotide Selection Checkpoints in the
Reaction Pathway of DNA Polymerase I and Roles of Glu710 and Tyr766 |
title_full | Prechemistry Nucleotide Selection Checkpoints in the
Reaction Pathway of DNA Polymerase I and Roles of Glu710 and Tyr766 |
title_fullStr | Prechemistry Nucleotide Selection Checkpoints in the
Reaction Pathway of DNA Polymerase I and Roles of Glu710 and Tyr766 |
title_full_unstemmed | Prechemistry Nucleotide Selection Checkpoints in the
Reaction Pathway of DNA Polymerase I and Roles of Glu710 and Tyr766 |
title_short | Prechemistry Nucleotide Selection Checkpoints in the
Reaction Pathway of DNA Polymerase I and Roles of Glu710 and Tyr766 |
title_sort | prechemistry nucleotide selection checkpoints in the
reaction pathway of dna polymerase i and roles of glu710 and tyr766 |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3770053/ https://www.ncbi.nlm.nih.gov/pubmed/23937394 http://dx.doi.org/10.1021/bi400837k |
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