Unwinding of a Herpes Simplex Virus Type 1 Origin of Replication (Ori _S ) by a Complex of the Viral Origin Binding Protein and the Single-Stranded DNA Binding Protein

Abstract

A herpes simplex virus type 1 (HSV-1) Ori S analogue in which the A؉T sequence linking the box I and II elements was replaced by two single-stranded oligo(dT)s is unwound by the UL9 protein-ICP8 complex. Unwinding of wild-type Ori S by the UL9 protein-ICP8 complex was also observed under conditions which destabilize the A؉T sequence. These experiments support a model for the unwinding of Ori S in which destabilization of the A؉T sequence can generate a single-stranded DNA binding site for ICP8, which then associates with the UL9 protein bound to boxes I and II to promote the bidirectional unwinding of Ori S .Herpes simplex virus type 1 (HSV-1) encodes a 94-kDa origin binding protein, the product of the UL9 gene, which is essential for HSV-1 DNA replication (5, 10). The origin binding protein (UL9 protein) is a homodimer that binds the two inverted pentanucleotide repeats, boxes I and II of the HSV-1 origin of replication, Ori S . Boxes I and II are linked by an AϩT-rich sequence of 18 nucleotide residues (5). In addition to its origin binding activity, the UL9 protein possesses DNAdependent ATPase and 3Ј-5Ј helicase activities (3, 6). Despite its helicase activity, we have been unable to detect the unwinding of duplex DNA containing Ori S (9).Previous studies have shown that a complex of the UL9 protein and the HSV-1-encoded single-stranded DNA binding protein, ICP8, can efficiently unwind a duplex box I if it possesses a 3Ј single-stranded tail at least 18 nucleotides in length, positioned downstream of box I (8). These findings suggested a model for the unwinding of Ori S in which a complex of the UL9 protein bound to boxes I and II and ICP8 bound to single-stranded DNA generated at the AϩT-rich linker, possibly as a consequence of transcription (4), unwinds the origin of DNA replication to provide access to the replication machinery, thereby permitting the initiation of DNA replication.To test this model, we have investigated the ability of the UL9 protein-ICP8 complex to unwind an Ori S analogue in which the AϩT-rich linker has been replaced by two single strands each consisting of 18 deoxythymidylate residues. We have found this Ori S analogue to be unwound by the complex. In addition, we have found that at temperatures that would be expected to destabilize the AϩT-rich linker, some unwinding of wild-type Ori S can be observed. Ori S bearing mutations in boxes I and II which inhibit binding of UL9 protein could not be unwound under these conditions. These findings support the model in which destabilization of the AϩT-rich linker to provide a binding site for ICP8 represents the initial event in the UL9 protein-promoted unwinding of Ori S .The structure of the duplex oligonucleotides used are shown in Fig. 1. The single-stranded oligonucleotides were purchased from Operon Technologies, Inc. They were purified prior to use by 16% polyacrylamide electrophoresis under denaturing conditions. The concentration of single-stranded oligonucleotides was determined spectrophotometrically assuming that 1 absorbance uni...

Origin-specific unwinding of herpes simplex virus 1 DNA by the viral UL9 and ICP8 proteins: Visualization of a specific preunwinding complex

“…The recent work of Lee and Lehman (10, 37) and He and Lehman (38,39) with model DNAs further supports the model in which supertwisting drives the melting of the A͞T-rich spacer. In Lee and Lehman (10,37), it was found that ICP8 would promote the unwinding of a short oriS-containing duplex if it either contained a ss tail or if the A͞T-rich spacer was unpaired.…”

“…In the presence of ICP8, UL9 protein will open a DNA-containing box I and a 3Ј-ssDNA tail located on the side oriented toward the A͞T-rich spacer (37). It will also open oriS constructs in which the A͞T-rich spacer is replaced by a ss bubble formed from two ss oligo(dT) segments located between boxes I and II (38) or wild-type oriS (39). Together, these experiments suggest a pathway of origin activation that involves a specific interaction between UL9 and ICP8 proteins, leading to a conformational change in the origin that facilitates more extensive unwinding.…”

Dual Functions of Single-stranded DNA-binding Protein in Helicase Loading at the Bacteriophage T4 DNA Replication Fork

“…Thus, protein-protein interactions between ICP8 and UL9 confer new properties upon the helicase, as is the case for gp32-dda interactions. The UL9-ICP8 complex has been implicated in the unwinding of HSV-1 origins of replication (51,52), suggesting a mechanism of action similar to the one we have proposed for dda-gp32 complex translocation at T4 replication forks (Fig. 7).…”

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

“…This assay, which can monitor the continuous unwinding of Ori s , has revealed that the initial phase of unwinding of Ori s consists of the UL9 protein and ICP8-dependent, but ATP-independent unwinding of the A ϩ T-rich sequence linking the two inverted repeats, boxes I and II. On addition of ATP, the UL9 protein-ICP8 complex can then promote the bidirectional unwinding of the entire Ori s sequence (3,4).…”