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J. Biol. Chem., Vol. 279, Issue 27, 28375-28386, July 2, 2004

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Human Kaposi's Sarcoma Herpesvirus Processivity Factor-8 Functions as a Dimer in DNA Synthesis^*

Xulin Chen, Kai Lin¶||, and Robert P. Ricciardi¶**

From the Departments of Microbiology, School of Dental Medicine and ^¶Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

In the absence of other proteins, the DNA polymerase (Pol-8) of Kaposi's sarcoma herpesvirus incorporates only several nucleotides from a primer template. However, association with the Kaposi's sarcoma herpesvirus processivity factor (PF-8) enables Pol-8 to incorporate thousands of nucleotides. Unlike the well described sliding clamp processivity factors, eukaryotic proliferating cell nuclear antigen and Escherichia coli -subunit, PF-8 and other herpesvirus processivity factors do not require a clamp loader or ATP to bind to template DNA. To begin to understand the mechanism used by PF-8 to achieve processivity, we have now purified PF-8 and demonstrated that it is a dimer both in solution and on the DNA. Mutational analysis of the PF-8 protein (396R) indicates that residues between 277 and 304 as well as the N-terminal 21 amino acids are required for dimerization. The results further correlate PF-8 dimerization with binding to Pol-8 and stabilizing Pol-8 on primer template. Notably, although removal of only 26 residues from the C terminus of PF-8 does not affect its ability to form dimers on DNA or to bind Pol-8, only short DNA chains (<100 nucleotides) are synthesized. This indicates that full-length PF-8 is necessary to enable Pol-8 to incorporate thousands of nucleotides. Interestingly, cross-linking of the processivity factor UL44 of cytomegalovirus reveals that it is a dimer in solution also.

Received for publication, January 5, 2004 , and in revised form, April 2, 2004.

^* This work was supported by National Cancer Institute research Grant CA80602 (to R. P. R.) and University of Pennsylvania Center for AIDS Research Grant P30 AI45008. 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.

Present address: Fox Chase Cancer Center, Philadelphia, PA 19111.

^|| Present address: Vertex Pharmaceuticals Inc., Cambridge, MA 02139.

^** To whom correspondence should be addressed. Tel.: 215-898-3905; Fax: 215-898-8385; E-mail: ricciardi{at}biochem.dental.upenn.edu.

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