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Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta
Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome. Saccharomyces cerevisiae DNA polymerase η (yPolη), a Y-family DNA polymerase, is known to efficiently bypass DNA lesions (e.g., pyrimidine dimers) in vivo. Using pre-s...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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SAGE-Hindawi Access to Research
2010
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925389/ https://www.ncbi.nlm.nih.gov/pubmed/20798853 http://dx.doi.org/10.4061/2010/871939 |
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author | Brown, Jessica A. Zhang, Likui Sherrer, Shanen M. Taylor, John-Stephen Burgers, Peter M. J. Suo, Zucai |
author_facet | Brown, Jessica A. Zhang, Likui Sherrer, Shanen M. Taylor, John-Stephen Burgers, Peter M. J. Suo, Zucai |
author_sort | Brown, Jessica A. |
collection | PubMed |
description | Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome. Saccharomyces cerevisiae DNA polymerase η (yPolη), a Y-family DNA polymerase, is known to efficiently bypass DNA lesions (e.g., pyrimidine dimers) in vivo. Using pre-steady-state kinetic methods, we examined both full-length and a truncated version of yPolη which contains only the polymerase domain. In the absence of yPolη's C-terminal residues 514–632, the DNA binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold. Thus, the C-terminus of yPolη may interact with DNA and slightly alter the conformation of the polymerase domain during catalysis. In general, yPolη discriminated between a correct and incorrect nucleotide more during the incorporation step (50-fold on average) than the ground-state binding step (18-fold on average). Blunt-end additions of dATP or pyrene nucleotide 5′-triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides. |
format | Text |
id | pubmed-2925389 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | SAGE-Hindawi Access to Research |
record_format | MEDLINE/PubMed |
spelling | pubmed-29253892010-08-26 Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta Brown, Jessica A. Zhang, Likui Sherrer, Shanen M. Taylor, John-Stephen Burgers, Peter M. J. Suo, Zucai J Nucleic Acids Research Article Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome. Saccharomyces cerevisiae DNA polymerase η (yPolη), a Y-family DNA polymerase, is known to efficiently bypass DNA lesions (e.g., pyrimidine dimers) in vivo. Using pre-steady-state kinetic methods, we examined both full-length and a truncated version of yPolη which contains only the polymerase domain. In the absence of yPolη's C-terminal residues 514–632, the DNA binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold. Thus, the C-terminus of yPolη may interact with DNA and slightly alter the conformation of the polymerase domain during catalysis. In general, yPolη discriminated between a correct and incorrect nucleotide more during the incorporation step (50-fold on average) than the ground-state binding step (18-fold on average). Blunt-end additions of dATP or pyrene nucleotide 5′-triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides. SAGE-Hindawi Access to Research 2010-07-25 /pmc/articles/PMC2925389/ /pubmed/20798853 http://dx.doi.org/10.4061/2010/871939 Text en Copyright © 2010 Jessica A. Brown et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Brown, Jessica A. Zhang, Likui Sherrer, Shanen M. Taylor, John-Stephen Burgers, Peter M. J. Suo, Zucai Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta |
title | Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta |
title_full | Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta |
title_fullStr | Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta |
title_full_unstemmed | Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta |
title_short | Pre-Steady-State Kinetic Analysis of Truncated and Full-Length Saccharomyces cerevisiae DNA Polymerase Eta |
title_sort | pre-steady-state kinetic analysis of truncated and full-length saccharomyces cerevisiae dna polymerase eta |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925389/ https://www.ncbi.nlm.nih.gov/pubmed/20798853 http://dx.doi.org/10.4061/2010/871939 |
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