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Nucleic Acids Research Advance Access originally published online on April 20, 2009
Nucleic Acids Research 2009 37(11):3774-3787; doi:10.1093/nar/gkp238
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Nucleic Acids Research, 2009, Vol. 37, No. 11 3774-3787
© 2009 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 Acids Enzymes

Low-fidelity DNA synthesis by the L979F mutator derivative of Saccharomyces cerevisiae DNA polymerase {zeta}

Jana E. Stone1, Grace E. Kissling2, Scott A. Lujan1, Igor B. Rogozin3, Carrie M. Stith4, Peter M. J. Burgers4 and Thomas A. Kunkel1,*

1Laboratory of Molecular Genetics and Laboratory of Structural Biology, 2Biostatistics Branch, National Institute of Environmental Health Sciences Research, NIH, DHHS, Research Triangle Park, NC 27709, 3National Center for Biotechnology Information, National Library of Medicine, NIH, DHHS, Bethesda, MD 20894 and 4Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA

*To whom correspondence should be addressed. Tel: +1 919 541 2644; Fax: +1 919 541 7613; Email: kunkel{at}niehs.nih.gov

Received February 11, 2009. Revised March 26, 2009. Accepted March 30, 2009.

To probe Pol {zeta} functions in vivo via its error signature, here we report the properties of Saccharomyces cerevisiae Pol {zeta} in which phenyalanine was substituted for the conserved Leu-979 in the catalytic (Rev3) subunit. We show that purified L979F Pol {zeta} is 30% as active as wild-type Pol {zeta} when replicating undamaged DNA. L979F Pol {zeta} shares with wild-type Pol {zeta} the ability to perform moderately processive DNA synthesis. When copying undamaged DNA, L979F Pol {zeta} is error-prone compared to wild-type Pol {zeta}, providing a biochemical rationale for the observed mutator phenotype of rev3-L979F yeast strains. Errors generated by L979F Pol {zeta} in vitro include single-base insertions, deletions and substitutions, with the highest error rates involving stable misincorporation of dAMP and dGMP. L979F Pol {zeta} also generates multiple errors in close proximity to each other. The frequency of these events far exceeds that expected for independent single changes, indicating that the first error increases the probability of additional errors within 10 nucleotides. Thus L979F Pol {zeta}, and perhaps wild-type Pol {zeta}, which also generates clustered mutations at a lower but significant rate, performs short patches of processive, error-prone DNA synthesis. This may explain the origin of some multiple clustered mutations observed in vivo.


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