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

Abstract

Herpes simplex virus 1 contains three origins of replication; two copies of oriS and one of a similar sequence, oriL. Here, the combined action of multiple factors known or thought to influence the opening of oriS are examined. These include the viral originbinding protein, UL9, and single-strand binding protein ICP8, host cell topoisomerase I, and superhelicity of the DNA template. By using electron microscopy, it was observed that when ICP8 and UL9 proteins were added together to oriS-containing supertwisted DNA, a discrete preunwinding complex was formed at oriS on 40% of the molecules, which was shown by double immunolabeling electron microscopy to contain both proteins. This complex was relatively stable to extreme dilution. Addition of ATP led to the efficient unwinding of Ϸ50% of the DNA templates. Unwinding proceeded until the acquisition of a high level of positive supertwists in the remaining duplex DNA inhibited further unwinding. Addition of topoisomerase I allowed further unwinding, opening >1 kb of DNA around oriS.T he initiation of DNA replication for most genomes begins with the recognition of the origin by specific origin-binding proteins. Binding frequently is accompanied by a structural alteration in the DNA that promotes unwinding and entry of the proteins required to initiate DNA synthesis (1, 2). Some origin-binding proteins also exhibit helicase activity, which facilitates origin unwinding, and several form hexamers or double hexamers on the DNA, a structure typical of many helicases; examples include simian virus 40 T antigen (3-5) and the E1 protein of the papillomaviruses (6, 7). Herpes simplex virus 1 (HSV-1) UL9 protein provides origin recognition function but binds as a double dimer to the HSV-1 origins rather than as a hexamer (8-10).HSV-1 provides an excellent system for study of these early replication steps in a mammalian system. The HSV-1 genome encodes seven proteins required for origin-dependent DNA replication consisting of a DNA polymerase and its accessory protein, a heterotrimeric helicase-primase, a single-stranded (ss) DNAbinding protein, ICP8, and the origin-binding protein, UL9 protein (11-16). HSV-1 contains three functional origins of DNA replication. One, oriL, is present in the long unique segment of the genome, whereas the other highly homologous origin, oriS, is present twice in the repeat region flanking the short unique segment (17)(18)(19)(20). The minimal functional oriS sequence (79 bp) consists of a 45-bp inverted repeat containing a central A͞T-rich element flanked on each side by two high-affinity UL9 proteinbinding sites designated box I and box II. A third weaker UL9 protein-binding site, box III, is located adjacent to box I.ICP8 is the major ssDNA-binding protein coded by the HSV-1 genome. ICP8 stimulates the helicase activity of UL9 protein (21, 22) and binds to its C-terminal domain (23). The ICP8-UL9 protein interaction is stable in the presence of double-stranded DNA but not ssDNA likely due to the strong affinity of ICP8 for ssDNA (9,24).UL9 prot...

Kaposi's Sarcoma-Associated Herpesvirus ori - Lyt -Dependent DNA Replication: Involvement of Host Cellular Factors

“…In herpes simplex virus type 1 (HSV-1), the origin binding protein UL9 is proposed to carry out this activity. UL9 is known to possess ATPase and helicase activities in addition to its origin (8,26). Genetic analysis showed that the conserved helicase motifs in UL9 are essential both in vivo and in vitro for viral DNA replication (27,28).…”

Replication and Recombination of Herpes Simplex Virus DNA

“…The recognition sequence for OBP, TTCGCAC, is highly conserved among alphaherpesviruses but differs from its counterpart in roseoloviruses (20). OBP binds cooperatively to oriS, and the assembly of a stable complex containing two OBP dimers on oriS as well as ICP8 has been visualized by electron microscopy (23). OBP alone can destabilize the AT-rich spacer sequence (24).…”

“…OBP alone can destabilize the AT-rich spacer sequence (24). In the presence of ATP, the preinitiation complex will proceed to unwind DNA and generate stretches of ϳ300-bp single-stranded DNA (ssDNA) covered with ICP8 within 15 min on linear DNA (23).…”

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping

“…The unwinding of the DNA is inherent to the function of helicases such as RNA polymerase (9), the mismatch repair MutS proteins (10) and HSV-1 UL9 protein (11,12), and hence these helicases must track the DNA. However, in the case of restriction enzymes (13) and the chromatin remodelling Snf2 proteins (14), there is no functional requirement to unwind the DNA.…”