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J. Biol. Chem., Vol. 279, Issue 21, 21957-21965, May 21, 2004

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Role of Protein-Protein Interactions during Herpes Simplex Virus Type 1 Recombination-dependent Replication^*

Amitabh V. Nimonkar and Paul E. Boehmer

From the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101-6129

Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201–10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.

Received for publication, January 26, 2004 , and in revised form, March 15, 2004.

^* This work was supported by Grant GM62643 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.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Miami School of Medicine, P. O. Box 016129, Miami, FL 33101-6129. Tel.: 305-243-2934; Fax: 305-243-3955; E-mail: pboehmer{at}molbio.med.miami.edu.

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				P. E. Boehmer RNA binding and R-loop formation by the herpes simplex virus type-1 single-stranded DNA-binding protein (ICP8) Nucleic Acids Res., August 25, 2004; 32(15): 4576 - 4584. [Abstract] [Full Text] [PDF]