The UL8 subunit of the heterotrimeric herpes simplex virus type 1 helicase-primase is required for the unwinding of single strand DNA-binding protein (ICP8)-coated DNA substrates.
The Herpes simplex virus type 1 primosome consists of three subunits that are the products of the UL5, UL8, and UL52 genes. The heterotrimeric enzyme has DNA-dependent ATPase, helicase, and primase activities. Earlier studies show that a subassembly consisting of the UL5 and UL52 gene products was indistinguishable from the heterotrimeric enzyme in its helicase and primase activities. We demonstrate here that the UL8 protein is required for the helicase activity of the UL5/52 subassembly on long duplex DNA substrates (>30 nucleotides) with a single-stranded DNA loading site fully coated with the virus-encoded single strand DNA binding protein, ICP8. The Escherichia coli single strand DNA binding protein cannot substitute for ICP8, suggesting a specific physical interaction between ICP8 and the UL8 protein. Surface plasmon resonance measurements demonstrated an interaction between ICP8 and the UL5/52/8 heterotrimer but not with the UL5/52 subassembly or the UL8 protein alone. At a subsaturating level of ICP8, the UL5/52 subassembly does show helicase activity, suggesting that the subassembly can bind to single-stranded DNA but not to ICP8-coated DNA.
Falkenberg
M
M
Departments of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307, USA.
Bushnell
D A
DA
Elias
P
P
Lehman
I R
IR
D000818:Animals; D002460:Cell Line; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D011487:Protein Conformation; D012031:Refractometry; D018411:Spodoptera; D013379:Substrate Specificity; D013499:Surface Properties; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D014764:Viral Proteins; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases