Structural elements required for the cooperative binding of the herpes simplex virus origin binding protein to oriS reside in the N-terminal part of the protein.

The origin binding protein (OBP) of herpes simplex virus type 1 is required to activate a viral origin of replication in vivo. We have used intact OBP as well as a truncated form of the protein expressed in Escherichia coli to investigate the protein-protein interactions, as well as the protein-DNA interactions involved in the formation of a nucleoprotein complex at a viral origin of replication (oriS) in vitro. The salient findings demonstrate that the N-terminal part of OBP is required for the cooperative binding of OBP to three sites (boxes I, II, and III) within oriS. A detailed model for the interaction of OBP with the viral origins of replication oriS and oriL is presented.     

Cooperative interactions between replication origin-bound molecules of herpes simplex virus origin-binding protein are mediated via the amino terminus of the protein.

The virally encoded origin binding protein (OBP) of herpes simplex virus (HSV) is required for viral DNA synthesis. OBP binds at the replication origin to initimultienzyme replication complex (Challberg, M. D., and Kelly, T. J. (1989) Annu Rev. Biochem. 58, 671-717), OBP binds to two sites at the replication origin. The sequence-specific interaction of OBP with each binding site is localized to the major groove, and in both HSV origins the two interaction surfaces are in phase, aligned on the same face of the helix (Hazuda, D. J., Perry, H. C., Naylor, A. M., and McClements, W. L. (1991) J. Biol. Chem. 261, 24621-24625). Using native gel electrophoresis, we now demonstrate that OBP binding to the origin is highly cooperative and that cooperativity requires the putative NH2-terminal leucine zipper. Neither the phase nor orientation of the binding sites affect cooperativity, suggesting that the interaction promotes wrapping of origin DNA around the OBP multimer. A comparison of OBP DNase I footprints with the DNase I footprints of a truncated protein defective in cooperativity demonstrates that the interaction between OBPs bound at sites I and II affects the conformation of the intervening DNA, particularly when the phase or orientation of the two sites is different from wild type. OBP may elicit a unique nucleoprotein structure which facilitates unwinding of the origin and/or assembly of the replication complex. We also demonstrate that OBP can exchange binding sites, forming interduplex complexes. This property may be important for reinitiation of DNA replication.     

ATP-dependent unwinding of a minimal origin of DNA replication by the origin-binding protein and the single-strand DNA-binding protein ICP8 from herpes simplex virus type I

The Herpes simplex virus type I origin-binding protein, OBP, is encoded by the UL9 gene. OBP binds the origin of DNA replication, oriS, in a cooperative and sequence-specific manner. OBP is also an ATP-dependent DNA helicase. We have recently shown that single-stranded oriS folds into a unique and evolutionarily conserved conformation, oriS*, which is stably bound by OBP. OriS* contains a stable hairpin formed by complementary base pairing between box I and box III in oriS. Here we show that OBP, in the presence of the single-stranded DNA-binding protein ICP8, can convert an 80-base pair double-stranded minimal oriS fragment to oriS* and form an OBP-oriS* complex. The formation of an OBP-oriS* complex requires hydrolysable ATP. We also demonstrate that OBP in the presence of ICP8 and ATP promotes slow but specific and complete unwinding of duplex minimal oriS. The possibility that the OBP-oriS* complex may serve as an assembly site for the herpes virus replisome is discussed.     

Characterization of the herpes simplex virus origin binding protein interaction with OriS.

The origin binding protein (OBP) of herpes simplex virus (HSV), which is essential for viral DNA replication, binds specifically to sequences within the viral replication origin(s) (for a review, see Challberg, M.D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717). Using either a COOH-terminal OBP protein A fusion or the full-length protein, each expressed in Escherichia coli, we investigated the interaction of OBP with one HSV origin, OriS. Binding of OBP to a set of binding site variant sequences demonstrates that the 10-base pair sequence, 5' CGTTCGCACT 3', comprises the OBP-binding site. This sequence must be presented in the context of at least 15 total base pairs for high affinity binding, Ka = approximately 0.3 nM. Single base pair mutations in the central CGC sequence lower the affinity by several orders of magnitude, whereas a substitution at any of the other seven positions reduces the affinity by 10-fold or less. OBP binds with high affinity to duplex DNA containing mismatched base pairs. This property is exploited to analyze OBP binding to DNA heteroduplexes containing singly substituted mutant and wild-type DNA strands. For positions 2, 3, 5, 6, 7, 8, and 9, substitutions are tolerated on one or the other DNA strand, indicating that base-mediated interactions are limited to one base of each pair. For both Boxes I and II, these interactions are localized to one face of the DNA helix, forming a recognition surface in the major groove. In OriS, the 31 base pairs which separate Boxes I and II orient the two interaction surfaces to the same side of the DNA.     

Targeting Holliday junctions by origin DNA-binding protein of herpes simplex virus type 1

In the present paper, the interactions of the origin binding protein (OBP) of herpes simplex virus type 1 (HSV1) with synthetic four-way Holliday junctions (HJs) were studied using electrophoresis mobility shift assay and the FRET method and compared with the interactions of the protein with duplex and single-stranded DNAs. It has been found that OBP exhibits a strong preference for binding to four-way and three-way DNA junctions and possesses much lower affinities to duplex and single-stranded DNAs. The protein forms three types of complexes with HJs. It forms complexes I and II which are reminiscent of the tetramer and octamer complexes with four-way junction of HJ-specific protein RuvA of Escherichia coli. The binding approaches saturation level when two OBP dimers are bound per junction. In the presence of Mg²⁺ ions (≥2 mM) OBP also interacts with HJ in the stacked arm form (complex III). In the presence of 5 mM ATP and 10 mM Mg²⁺ ions OBP catalyzes processing of the HJ in which one of the annealed oligonucleotides has a 3′-terminal tail containing 20 unpaired thymine residues. The observed preference of OBP for binding to the four-way DNA junctions provides a basis for suggestion that OBP induces large DNA structural changes upon binding to Box I and Box II sites in OriS. These changes involve the bending and partial melting of the DNA at A+T-rich spacer and also include the formation of HJ containing Box I and Box II inverted repeats and flanking DNA sequences.     

Analysis of the herpes simplex virus type 1 OriS sequence: mapping of functional domains.

The herpes simplex virus type 1 (HSV-1) OriS region resides within a 90-bp sequence that contains two binding sites for the origin-binding protein (OBP), designated sites I and II. A third presumptive OBP-binding site (III) within OriS has strong sequence similarity to sites I and II, but no sequence-specific OBP binding has yet been demonstrated at this site. We have generated mutations in sites I, II, and III and determined their replication efficiencies in a transient in vivo assay in the presence of a helper virus. Mutations in any one of the sites reduced DNA replication significantly. To study the role of OriS sequence elements in site I and the presumptive site III in DNA replication, we have also generated a series of mutations that span from site I across the presumptive binding site III. These mutants were tested for their ability to replicate and for the ability to bind OBP by using gel shift analyses. The results indicate that mutations across site I drastically reduce DNA replication. Triple-base-pair substitution mutations that fall within the crucial OBP-binding domain, 5'-YGYTCGCACT-3' (where Y represents C or T), show a reduced level of OBP binding and DNA replication. Substitution mutations in site I that are outside this crucial binding sequence show a more detrimental effect on DNA replication than on OBP binding. This suggests that these sequences are required for initiation of DNA replication but are not critical for OBP binding. Mutations across the presumptive OBP-binding site III also resulted in a loss in efficiency of DNA replication. These mutations influenced OBP binding to OriS in gel shift assays, even though the mutated sequences are not contained within known OBP-binding sites. Replacement of the wild-type site III with a perfect OBP-binding site I results in a drastic reduction of DNA replication. Thus, our DNA replication assays and in vitro DNA-binding studies suggest that the binding of the origin sequence by OBP is not the only determining factor for initiation of DNA replication in vivo.     

Characterization of major recognition sequences for a herpes simplex virus type 1 origin-binding protein.

To investigate early initiation events in the replication of herpes simplex virus type 1, we analyzed interactions of proteins from infected cell extracts with the small origin of herpes simplex virus type 1 (oris1). Using the mobility shift assay, we detected two origin-specific binding interactions. We characterized the more prominent interaction on both strands of the DNA duplex with DNase I protection and methylation interference assays. Protein binding protects 17 bases of DNA on each strand from DNase I. These sequences are located at the left end of the central palindrome and are shifted four bases relative to one another. On the basis of the DNase protection pattern, we believe this protein to be related to the origin-binding protein defined by Elias et al. (P. Elias, M.E. O'Donnell, E.S. Mocarski, and I.R. Lehman, Proc. Natl. Acad. Sci. 83:6322-6326, 1986). Our DNase I footprint shows both strong and weak areas of protection. The regions strongly protected from DNase I align with the essential contact residues identified by interference footprinting. Methylation interference defines a small binding domain of 8 base pairs: 5'-GTTCGCAC-3'/3'-CAAGCGTG-5'. This recognition sequence contains two inverted 5'-GT(T/G)CG-3' repeats which share a 2-base overlap; thus, the origin-binding protein probably binds to the inverted repeats as a dimer.     

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

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).     

Cellular protein interactions with herpes simplex virus type 1 oriS.

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.     

Sequence requirements for interaction of human herpesvirus 7 origin binding protein with the origin of lytic replication

As do human herpesvirus 6 variants A and B (HHV-6A and -6B), HHV-7 encodes a homolog of the alphaherpesvirus origin binding protein (OBP), which binds at sites in the origin of lytic replication (oriLyt) to initiate DNA replication. In this study, we sought to characterize the interaction of the HHV-7 OBP (OBP(H7)) with its cognate sites in the 600-bp HHV-7 oriLyt. We expressed the carboxyl-terminal domain of OBP(H7) and found that amino acids 484 to 787 of OBP(H7) were sufficient for DNA binding activity by electrophoretic mobility shift analysis. OBP(H7) has one high-affinity binding site (OBP-2) located on one flank of an AT-rich spacer element and a low-affinity site (OBP-1) on the other. This is in contrast to the HHV-6B OBP (OBP(H6B)), which binds with similar affinity to its two cognate OBP sites in the HHV-6B oriLyt. The minimal recognition element of the OBP-2 site was mapped to a 14-bp sequence. The OBP(H7) consensus recognition sequence of the 9-bp core, BRTYCWCCT (where B is a T, G, or C; R is a G or A; Y is a T or C; and W is a T or A), overlaps with the OBP(H6B) consensus YGWYCWCCY and establishes YCWCC as the roseolovirus OBP core recognition sequence. Heteroduplex analysis suggests that OBP(H7) interacts along one face of the DNA helix, with the major groove, as do OBP(H6B) and herpes simplex virus type 1 OBP. Together, these results illustrate both conserved and divergent DNA binding properties between OBP(H7) and OBP(H6B).     

Functional properties of the herpes simplex virus type I origin-binding protein are controlled by precise interactions with the activated form of the origin of DNA replication

The herpes simplex virus, type I origin-binding protein, OBP, is a superfamily II DNA helicase encoded by the UL9 gene. OBP binds in a sequence-specific and cooperative way to the viral origin of replication oriS. OBP may unwind partially and introduce a hairpin into the double-stranded origin of replication. The formation of the novel conformation referred to as oriS* also requires the single-stranded DNA-binding protein, ICP8, and ATP hydrolysis. OBP forms a stable complex with oriS*. The hairpin in oriS* provides a site for sequence-specific attachment, and a single-stranded region triggers ATP hydrolysis. Here we use Escherichia coli exonuclease I to map the binding of the C-terminal domain of OBP to the hairpin and the helicase domains to the single-stranded tail. The helicase domains cover a stretch of 23 nucleotides of single-stranded DNA. Using streptavidin-coated magnetic beads, we show that OBP may bind two copies of double-stranded DNA (one biotin-labeled and the other one radioactively labeled) but only one copy of oriS*. It is the length of the single-stranded tail that determines the stoichiometry of OBP.DNA complexes. OBP interacts with the bases of the single-stranded tail, and ATP hydrolysis is triggered by position-specific interactions between OBP and bases in the single-stranded tail of oriS*.     

A novel conformation of the herpes simplex virus origin of DNA replication recognized by the origin binding protein

The Herpes simplex virus type I origin binding protein (OBP) is a sequence-specific DNA-binding protein and a dimeric DNA helicase encoded by the UL9 gene. It is required for the activation of the viral origin of DNA replication oriS. Here we demonstrate that the linear double-stranded form of oriS can be converted by heat treatment to a stable novel conformation referred to as oriS*. Studies using S1 nuclease suggest that oriS* consists of a central hairpin with an AT-rich sequence in the loop. Single-stranded oligonucleotides corresponding to the upper strand of oriS can adopt the same structure. OBP forms a stable complex with oriS*. We have identified structural features of oriS* recognized by OBP. The central oriS palindrome as well as sequences at the 5' side of the oriS palindrome were required for complex formation. Importantly, we found that mutations that have been shown to reduce oriS-dependent DNA replication also reduce the formation of the OBP-oriS* complex. We suggest that oriS* serves as an intermediate in the initiation of DNA replication providing the initiator protein with structural information for a selective and efficient assembly of the viral replication machinery.     

Mutations in a herpes simplex virus type 1 origin that inhibit interaction with origin-binding protein also inhibit DNA replication.

The herpes simplex virus type 1 genome contains three origins of replication: OriL and a diploid OriS. The origin-binding protein, the product of the UL9 gene, interacts with two sites within OriS, box I and box II. A third site, box III, which is homologous to boxes I and II, may also be a binding site for the origin-binding protein. Mutations in these three sites significantly reduce OriS-directed plasmid replication measured in transient replication assays. The reduction in replication efficiency of the mutants correlates well with the decrease in the ability to bind to the origin-binding protein, as determined by Elias et al. (P. Elias, C. M. Gustafsson, and O. Hammarsten, J. Biol. Chem. 265: 17167-17173, 1990). The effect of multiple mutations in boxes I, II, and III on plasmid replication suggests that there are multiple binding sites in OriS for the origin-binding protein. These studies indicate that proper interaction of the origin-binding protein with the OriS sequence is essential for OriS-directed DNA replication.     

Phosphorylation of the herpes simplex virus type 1 origin binding protein

The herpes simplex virus type 1 (HSV-1) origin binding protein (OBP), the product of the UL9 gene, is one of seven HSV-encoded proteins required for viral DNA replication. OBP performs multiple functions characteristic of a DNA replication initiator protein, including origin-specific DNA binding and ATPase and helicase activities, as well as the ability to interact with viral and cellular proteins involved in DNA replication. Replication initiator proteins in other systems, including those of other DNA viruses, are known to be regulated by phosphorylation; however, the role of phosphorylation in OBP function has been difficult to assess due to the low level of OBP expression in HSV-infected cells. Using a metabolic labeling and immunoprecipitation approach, we obtained evidence that OBP is phosphorylated during HSV-1 infection. Kinetic analysis of metabolically labeled cells indicated that the levels of OBP expression and phosphorylation increased at approximately 4 h postinfection. Notably, when expressed from a transfected plasmid, a recombinant baculovirus, or a recombinant adenovirus (AdOBP), OBP was phosphorylated minimally, if at all. In contrast, superinfection of AdOBP-infected cells with an OBP-null mutant virus increased the level of OBP phosphorylation approximately threefold, suggesting that HSV-encoded viral or HSV-induced cellular factors enhance the level of OBP phosphorylation. Using HSV mutants inhibited at sequential stages of the viral life cycle, we demonstrated that this increase in OBP phosphorylation is dependent on early protein synthesis and is independent of viral DNA replication. Based on gel mobility shift assays, phosphorylation does not appear to affect the ability of OBP to bind to the HSV origins.     

Cloning, sequencing, and functional analysis of a Marek''s disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.     

Interaction between the replication origin and the initiator protein of the filamentous phage f1. Binding occurs in two steps

The replication initiator protein of bacteriophage f1 (gene II protein) binds to the phage origin and forms two complexes that are separable by polyacrylamide gel electrophoresis. Complex I is formed at low gene II protein concentrations, and shows protection from DNase I of about 25 base-pairs (from position +2 to +28 relative to the nicking site) at the center of the minimal origin sequence. Complex II is produced at higher concentrations of the protein, and has about 40 base-pairs (from -7 to +33) protected. On the basis of gel mobility, complex II appears to contain twice the amount of gene II protein as does complex I. The 40 base-pair sequence protected in complex II corresponds to the minimal origin sequence as determined by in-vivo analyses. The central 15 base-pair sequence (from +6 to +20) of the minimal origin consists of two repeats in inverted orientation. This sequence, when cloned into a plasmid, can form complex I, but not complex II. We call this 15 base-pair element the core binding sequence for gene II protein. Methylation interference with the formation of complex I by the wild-type origin indicates that gene II protein contacts six guanine residues located in a symmetric configuration within the core binding sequence. Formation of complex II requires, in addition to the core binding sequence, the adjacent ten base-pair sequence on the right containing a third homologous repeat. A methylation interference experiment performed on complex II indicates that gene II protein interacts homologously with the three repeats. In complex II, gene II protein protects from DNase I digestion not only ten base-pairs on the right but also ten base-pairs on the left of the sequence that is protected in complex I. Footprint analyses of various deletion mutants indicate that the left-most ten base-pairs are protected regardless of their sequence. The site of nicking by gene II protein is located within this region. A model is presented for the binding reaction involving both protein-DNA and protein-protein interactions.     

Binding sites of HeLa cell nuclear proteins on the upstream region of adenovirus type 5 E1A gene.

Twenty one binding sites of HeLa cell nuclear proteins were identified on the upstream region of adenovirus type 5 E1A gene using DNase I footprint assay. The proximal promoter region contained five binding sites that overlapped the cap site, TATA box, TATA-like sequence, CCAAT box, and -100 region relative to the E1A cap site(+1). The -190 region was a potential site for octamer-motif binding proteins, such as NFIII and OBP100. An upstream copy of the E1A enhancer element 1 was the site for a factor (E1A-F) with the binding specificity of XGGAYGT (X = A, C; Y = A, T). E1A-F factor also bound to three other sites, one of which coincided with the distal E1A enhancer element. The distal element also contained a potential site for ATF factor. The adenovirus minimal origin of DNA replication competed for DNA-protein complex formation on the CCAAT and TATA box region and the -190 region, suggesting that these regions interacted with a common or related factor.