An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding protein and single-strand DNA-binding protein.
Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Ori(s), by a complex of the HSV-1 origin binding protein (UL9 protein) and the HSV-1 single-strand DNA binding protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 protein binding sites, boxes I and II of Ori(s). P1 nuclease sensitivity of Ori(s) and single-strand DNA-dependent ATPase measurements of the UL9 protein indicate that, at 37 degrees C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 protein to boxes I and II then results in the formation of the UL9 protein-ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 protein-ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Ori(s) sequences.
Department of Biochemistry, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305-5307, USA.
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D000225:Adenine; D000255:Adenosine Triphosphate / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D018741:Replication Origin; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013941:Thymine; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1; D000255:Adenosine Triphosphate; D000225:Adenine; D013941:Thymine