The herpes simplex virus type 1 alkaline nuclease and single-stranded DNA binding protein mediate strand exchange in vitro.
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 Redalpha, 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 Redbeta 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 MgCl(2), 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.
Reuven
Nina Bacher
NB
Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06030-3205, USA.
Staire
Amy E
AE
Myers
Richard S
RS
Weller
Sandra K
SK
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases; D018411:Spodoptera; D014764:Viral Proteins / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D011994:Recombinant Proteins; D014764:Viral Proteins; D012260:Ribonucleases; C066964:viral alkaline nuclease