Abstract
Herpes simplex virus type-1 UL9 protein is a sequence-specific DNA-binding protein that recognizes elements in the viral origins of DNA replication and possesses DNA helicase activity. It forms an essential complex with its cognate single-strand DNA-binding protein, ICP8. The DNA helicase activity of the UL9 protein is greatly stimulated as a consequence of this interaction. A complex of these two proteins is thought to be responsible for unwinding the viral origins of DNA replication. The aim of this study was to identify the mechanism by which ICP8 stimulates the translocation of the UL9 protein along DNA. The data show that the association of the UL9 protein with DNA substrate is slow and that its dissociation from the DNA substrate is fast, suggesting that it is nonprocessive. ICP8 caused maximal stimulation of DNA unwinding activity at equimolar UL9 protein concentrations, indicating that the active species is a complex that contains UL9 protein and ICP8 in 1:1 ratio. ICP8 prevented dissociation of UL9 protein from the DNA substrate, suggesting that it increases its processivity. ICP8 specifically stimulated the DNA-dependent ATPase activity of the UL9 protein with DNA cofactors that allow translocation of UL9 protein and those with secondary structure. These data suggest that UL9 protein and ICP8 form a specific complex that translocates along DNA. Within this complex, ICP8 tethers the UL9 protein to the DNA substrate, thereby preventing its dissociation, and participates directly in the assimilation and stabilization of the unwound DNA strand, thus facilitating translocation of the complex through regions of duplex DNA.
Highlights
Herpes simplex virus type-1 (HSV-1)1 is a double-stranded DNA virus with a genome of ϳ152 kilobase pairs that contains three origins of DNA replication [1]
These results suggest that UL9 protein needs to assemble on the DNA substrate and that this association is slow and possibly rate-limiting
When UL9 protein was added to ICP8-coated DNA substrate, there was a slower rate of reaction (Fig. 1B)
Summary
Herpes simplex virus type-1 (HSV-1) is a double-stranded DNA virus with a genome of ϳ152 kilobase pairs that contains three origins of DNA replication [1]. Replication of origin-containing plasmids requires the action of seven viral gene products [2, 3] These seven gene products comprise a highly processive heterodimeric DNA polymerase (UL30/UL42 genes), a heterotrimeric DNA helicase-primase (UL5/UL8/UL52 genes), a single-strand DNA-binding protein (SSB) (UL29 gene), and an origin-binding protein (UL9 gene) (reviewed in Ref. 1). The ability of ICP8 to participate in multiple protein-protein interactions suggests that it fulfills several roles during viral DNA replication. It has been shown that disruption of the ICP8-UL9 protein complex by deletion of the 27 C-terminal amino acid of the UL9 protein greatly reduces origin-specific DNA replication [12]. The ICP8-UL9 protein complex promotes efficient unwinding of the HSV-1 origins of DNA replication [20, 21]. The results show that ICP8 increases the processivity of the UL9 protein, facilitating its translocation along DNA and through regions of secondary structure
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