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Nucleic Acids Research Advance Access originally published online on September 28, 2007
Nucleic Acids Research 2007 35(19):6588-6597; doi:10.1093/nar/gkm741
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Nucleic Acids Research, 2007, Vol. 35, No. 19 6588-6597
© 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

The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA

Olga Chilkova1, Peter Stenlund2, Isabelle Isoz1, Carrie M. Stith3, Pawel Grabowski1, Else-Britt Lundström1, Peter M. Burgers3 and Erik Johansson1,*

1Department of Medical Biochemistry and Biophysics, 2Department of Biochemistry, Umeå University, 901 87 Umeå, Sweden and 3Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, USA

*To whom correspondence should be addressed. Tel: +46 90 786 6638; Fax: +46 90 786 9795; Email: erik.johansson{at}medchem.umu.se

Received June 9, 2007. Revised September 6, 2007. Accepted September 6, 2007.

Saccharomyces cerevisiae DNA polymerase {delta} (Pol {delta}) and DNA polymerase {varepsilon} (Pol {varepsilon}) are replicative DNA polymerases at the replication fork. Both enzymes are stimulated by PCNA, although to different levels. To understand why and to explore the interaction with PCNA, we compared Pol {delta} and Pol {varepsilon} in physical interactions with PCNA and nucleic acids (with or without RPA), and in functional assays measuring activity and processivity. Using surface plasmon resonance technique, we show that Pol {varepsilon} has a high affinity for DNA, but a low affinity for PCNA. In contrast, Pol {delta} has a low affinity for DNA and a high affinity for PCNA. The true processivity of Pol {delta} and Pol {varepsilon} was measured for the first time in the presence of RPA, PCNA and RFC on single-stranded DNA. Remarkably, in the presence of PCNA, the processivity of Pol {delta} and Pol {varepsilon} on RPA-coated DNA is comparable. Finally, more PCNA molecules were found on the template after it was replicated by Pol {varepsilon} when compared to Pol {delta}. We conclude that Pol {varepsilon} and Pol {delta} exhibit comparable processivity, but are loaded on the primer-end via different mechanisms.

Present address: Peter Stenlund, Biopharmaceuticals Octapharma AB, 11275 Stockholm, Sweden

Pawel Grabowski, Department of Medical Biosciences, Umeå University, 901 87 Umeå, Sweden


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