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Journal of Virology, July 2003, p. 7425-7433, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7425-7433.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Herpes Simplex Virus Type 1 Alkaline Nuclease and Single-Stranded DNA Binding Protein Mediate Strand Exchange In Vitro

Nina Bacher Reuven,1 Amy E. Staire,1 Richard S. Myers,2 and Sandra K. Weller1*

Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06030-3205,1 Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101-61292

Received 2 January 2003/ Accepted 1 April 2003

The replication of herpes simplex virus type 1 (HSV-1) DNA is associated with a high degree of homologous recombination. While cellular enzymes may take part in mediating this recombination, we present evidence for an HSV-1-encoded recombinase activity. HSV-1 alkaline nuclease, encoded by the UL12 gene, is a 5'->3' exonuclease that shares homology with Red{alpha}, commonly known as {lambda} exonuclease, an exonuclease required for homologous recombination by bacteriophage lambda. The HSV-1 single-stranded DNA binding protein ICP8 is an essential protein for HSV DNA replication and possesses single-stranded DNA annealing activities like the Redß synaptase component of the phage lambda recombinase. Here we show that UL12 and ICP8 work together to effect strand exchange much like the Red system of lambda. Purified UL12 protein and ICP8 mediated the complete exchange between a 7.25-kb M13mp18 linear double-stranded DNA molecule and circular single-stranded M13 DNA, forming a gapped circle and a displaced strand as final products. The optimal conditions for strand exchange were 1 mM MgCl2, 40 mM NaCl, and pH 7.5. Stoichiometric amounts of ICP8 were required, and strand exchange did not depend on the nature of the double-stranded end. Nuclease-defective UL12 could not support this reaction. These data suggest that diverse DNA viruses appear to utilize an evolutionarily conserved recombination mechanism.

* Corresponding author. Mailing address: Department of Microbiology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3205. Phone: (860) 679-2310. Fax: (860) 679-1239. E-mail: weller{at}nso2.uchc.edu.

Journal of Virology, July 2003, p. 7425-7433, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7425-7433.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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