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J. Biol. Chem., Vol. 279, Issue 3, 1907-1915, January 16, 2004

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The Pol32 Subunit of DNA Polymerase Contains Separable Domains for Processive Replication and Proliferating Cell Nuclear Antigen (PCNA) Binding^*

Erik Johansson, Parie Garg, and Peter M. J. Burgers

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

We have carried out a domain analysis of POL32, the third subunit of Saccharomyces cerevisiae DNA polymerase (Pol ). Interactions with POL31, the second subunit of Pol , are specified by the amino-terminal 92 amino acids, whereas interactions with the replication clamp proliferating cell nuclear antigen (PCNA, POL30) reside at the extreme carboxyl-terminal region. Pol32 binding, in vivo and in vitro, to the large subunit of DNA polymerase , POL1, requires the carboxyl-proximal region of Pol32. The amino-terminal region of Pol32 is essential for damage-induced mutagenesis. However, the presence of its carboxyl-terminal PCNA-binding domain enhances the efficiency of mutagenesis, particularly at high loads of DNA damage. In vitro, in the absence of effector DNA, the PCNA-binding domain of Pol32 is essential for PCNA-Pol interactions. However, this domain has minimal importance for processive DNA synthesis by the ternary DNA-PCNA-Pol complex. Rather, processivity is determined by PCNA-binding domains located in the Pol3 and/or Pol31 subunits. Using diagnostic PCNA mutants, we show that during DNA synthesis the carboxyl-terminal domain of Pol32 interacts with the carboxyl-terminal region of PCNA, whereas interactions of the other subunit(s) of Pol localize largely to a hydrophobic pocket at the interdomain connector loop region of PCNA.

Received for publication, September 17, 2003 , and in revised form, October 30, 2003.

^* This work was supported in part by Grant GM58534 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a stipend from the Washington University-Umeå University Exchange program and a fellowship from the Swedish Cancer Society. Present address: Dept of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

To whom correspondence should be addressed. Tel.: 314-362-3872; Fax: 314-362-7183; E-mail: burgers{at}biochem.wustl.edu.

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