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Nucleic Acids Research Advance Access published online on April 22, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm132
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© 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.

Nucleic Acid Enzymes

Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase {varepsilon}

Zachary F. Pursell1, Isabelle Isoz2, Else-Britt Lundström2, Erik Johansson2 and Thomas A. Kunkel1,*

1Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA and 2Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87, Umeå, Sweden

*To whom correspondence should be addressed. Tel: +1-9195412644; Fax: +1-9195417613; Email: kunkel{at}niehs.nih.gov

Received January 18, 2007. Revised February 16, 2007. Accepted February 18, 2007.

To better understand the functions and fidelity of DNA polymerase {varepsilon} (Pol {varepsilon}), we report here on the fidelity of yeast Pol {varepsilon} 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 {varepsilon} synthesize DNA with high fidelity, but M644F Pol {varepsilon} 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 {varepsilon}. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol {alpha} (L868M/F) and Pol {delta} (L612M), these data indicate that the active site location occupied by Met644 in Pol {varepsilon} is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol {varepsilon} is distinct from that of L868M/F Pol {alpha} or L612M Pol {delta}, 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.


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