Catalysis of strand exchange by the HSV-1 UL12 and ICP8 proteins: potent ICP8 recombinase activity is revealed upon resection of dsDNA substrate by nuclease.
The replication of herpes simplex virus type 1 (HSV-1) is associated with a high degree of homologous recombination, which is likely to be mediated, in part, by HSV-1-encoded proteins. We have previously shown that the HSV-1 encoded ICP8 protein and alkaline nuclease UL12 are capable of catalyzing an in vitro strand-exchange reaction. Here, we show, by electron microscopy, that the products of the strand exchange reaction between linear double-stranded DNA and circular single-stranded DNA consist of the expected joint molecule forms: sigma, alpha, and gapped circles. Other exonucleases, such as lambda Red alpha, which, like UL12, digests 5'-3', as well as Escherichia coli exonuclease III (ExoIII), which digests 3'-5', could substitute for UL12 in the strand exchange reaction by providing a resected DNA end. ICP8 generated the same intermediates and strand exchange products when the double-stranded DNA substrate was preresected by any of the nucleases. Using substrates with large regions of non-homology we found that pairing by ICP8 could be initiated from the middle of a DNA molecule and did not require a homologous end. In this reaction, the resection of a DNA end by the nuclease is required to reveal homologous sequences capable of being paired by ICP8. This study further illustrates the complexity of the multi-functional ICP8 protein.
Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington 06030-3205, USA.
Nina B
NB
Reuven
Smaranda
S
Willcox
Jack D
JD
Griffith
Sandra K
SK
Weller
D004247:DNA / Q000378:metabolism* / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D014764:Viral Proteins / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000378:metabolism*; D014779:Virus Replication
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D014764:Viral Proteins; D015678:Viral Structural Proteins; D004247:DNA; D005090:Exodeoxyribonucleases