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Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε
To better understand the functions and fidelity of DNA polymerase ε (Pol ε), we report here on the fidelity of yeast Pol ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2007
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1888828/ https://www.ncbi.nlm.nih.gov/pubmed/17452367 http://dx.doi.org/10.1093/nar/gkm132 |
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author | Pursell, Zachary F. Isoz, Isabelle Lundström, Else-Britt Johansson, Erik Kunkel, Thomas A. |
author_facet | Pursell, Zachary F. Isoz, Isabelle Lundström, Else-Britt Johansson, Erik Kunkel, Thomas A. |
author_sort | Pursell, Zachary F. |
collection | PubMed |
description | To better understand the functions and fidelity of DNA polymerase ε (Pol ε), we report here on the fidelity of yeast Pol ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644W and M644L Pol ε synthesize DNA with high fidelity, but M644F Pol ε has reduced fidelity resulting from strongly increased misinsertion rates. When Msh6-dependent repair of replication errors is defective, the mutation rate of a pol2-M644F strain is 16-fold higher than that of a strain with wild-type Pol ε. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol α (L868M/F) and Pol δ (L612M), these data indicate that the active site location occupied by Met644 in Pol ε is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol ε is distinct from that of L868M/F Pol α or L612M Pol δ, implying that each polymerase has different active site geometry, and suggesting that these polymerase alleles may generate distinctive mutational signatures for probing functions in vivo. |
format | Text |
id | pubmed-1888828 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-18888282007-06-22 Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε Pursell, Zachary F. Isoz, Isabelle Lundström, Else-Britt Johansson, Erik Kunkel, Thomas A. Nucleic Acids Res Nucleic Acid Enzymes To better understand the functions and fidelity of DNA polymerase ε (Pol ε), we report here on the fidelity of yeast Pol ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644W and M644L Pol ε synthesize DNA with high fidelity, but M644F Pol ε has reduced fidelity resulting from strongly increased misinsertion rates. When Msh6-dependent repair of replication errors is defective, the mutation rate of a pol2-M644F strain is 16-fold higher than that of a strain with wild-type Pol ε. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol α (L868M/F) and Pol δ (L612M), these data indicate that the active site location occupied by Met644 in Pol ε is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol ε is distinct from that of L868M/F Pol α or L612M Pol δ, implying that each polymerase has different active site geometry, and suggesting that these polymerase alleles may generate distinctive mutational signatures for probing functions in vivo. Oxford University Press 2007-05 2007-04-22 /pmc/articles/PMC1888828/ /pubmed/17452367 http://dx.doi.org/10.1093/nar/gkm132 Text en © 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. |
spellingShingle | Nucleic Acid Enzymes Pursell, Zachary F. Isoz, Isabelle Lundström, Else-Britt Johansson, Erik Kunkel, Thomas A. Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε |
title | Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε |
title_full | Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε |
title_fullStr | Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε |
title_full_unstemmed | Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε |
title_short | Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase ε |
title_sort | regulation of b family dna polymerase fidelity by a conserved active site residue: characterization of m644w, m644l and m644f mutants of yeast dna polymerase ε |
topic | Nucleic Acid Enzymes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1888828/ https://www.ncbi.nlm.nih.gov/pubmed/17452367 http://dx.doi.org/10.1093/nar/gkm132 |
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