The herpes simplex virus (HSV) ()genome contains three cis-acting regions that function as origins of replication: two identical copies of a sequence called ori and one copy of a very closely related sequence called ori(3) . Although the mechanism by which DNA replication is initiated at these sequences is not yet known, there is considerable information regarding both the elements of the origin sequence that are important for replication and the proteins with which the origin interacts. Ori contains at least five functional domains: two high affinity binding sites (called site I and site II or box I and box II) for UL9, a virally encoded DNA-binding protein that is known to be essential for viral DNA replication(3, 4, 5, 6, 7, 8, 9, 10) ; an A/T-rich region; a sequence homologous to site I but with much lower affinity for UL9, called site III (or box III); and a binding site for an uncharacterized cellular protein(s) called OF-1(11) . Sites I and II are located on the arms of a 46-base pair palindrome and separated by the A/T-rich region. Nuclease protection, chemical modification, and saturation mutagenesis studies have shown that the high affinity recognition sequence for UL9 (site I) is contained in the 10-base pair sequence 5`-CGTTCGCACT(8, 9, 12) . Site II differs from this sequence at two positions, resulting in a reduced binding affinity for UL9 to about one-fifth that of site I (5, 8, 9) . Results of genetic studies have suggested that the binding of UL9 to the origin is important for viral DNA replication: mutations in ori that decrease the ability of UL9 to bind to site I or site II significantly decrease the efficiency of replication of the ori-containing plasmid in transient replication assays(13, 14) .

UL9 is comprised of 951 amino acids with a predicted molecular mass of 94 kDa and contains at least two functional domains: the carboxyl-terminal domain of 317 amino acids mediates sequence-specific DNA binding(1, 2, 7, 9, 15, 16, 17) , and the amino-terminal two-thirds of the protein mediates a DNA-dependent helicase activity, allowing UL9 to unwind partially duplex DNA of nonspecific sequence(18, 19, 20, 21) . In addition, this domain also appears to mediate dimerization and protein-protein interactions between UL9 molecules(22, 23) . UL9 has not been shown to unwind fully duplex DNA or to unwind preferentially origin-containing sequences(19, 24) . Nevertheless, by analogy with better characterized viral initiator proteins such as the simian virus 40 T antigen or the O protein (reviewed in 25), UL9 may initiate HSV DNA replication by binding to the origin and unwinding a local region of DNA to allow or direct the assembly of the HSV DNA replication machinery.

UL9 is a homodimer in solution (18, 19) and binds to ori in a cooperative manner at sites I and II(8, 22, 23) . The simplest models for the binding of UL9 to the origin, therefore, entail the binding of either one UL9 dimer to ori, with a monomer unit bound each at site I and site II, or two UL9 dimers bound to ori, one each to site I and site II. It is likely that a complete understanding of the early events of viral DNA replication will depend on an accurate determination of the binding stoichiometry between UL9 and the origin. Recently, two groups have used the carboxyl-terminal 317-amino acid DNA binding domain of UL9 in a gel shift assay to measure the binding stoichiometry between UL9 and the origin. The two groups, however, obtained opposing results: Martin et al.(1) reported that a monomer of UL9 binds to a single high affinity UL9 binding site; in contrast, Stabell and Olivo (2) concluded that a dimer of UL9 binds to a single high affinity site. These two results cannot be reconciled easily. The conclusions drawn from these gel shift assays, however, were inferential; both indirectly measured the binding stoichiometry of UL9 and ori. We have, therefore, directly measured the stoichiometry of binding between the carboxyl-terminal DNA binding domain of UL9 and the origin of replication. In this paper, we show that two monomer DNA binding domains of UL9 bind to a single high affinity UL9 binding site. Furthermore, in the context of the intact ori, four monomer UL9 binding sites are bound: two each to site I and site II.

EXPERIMENTAL PROCEDURES

Construction of Recombinant Baculovirus Expressing His-tagged t-UL9

Purification of H-Labeled His-tagged t-UL9

Determination of the Molar Extinction Coefficient of t-UL9

Preparation of DNA Substrates

Double-label Gel Shift Assays

RESULTS AND DISCUSSION

The DNA binding domain of UL9 (t-UL9) was used to determine the binding stoichiometry for UL9 and ori by using a double-label gel shift assay()(31, 32, 33, 34, 35, 36, 37) . The truncated protein rather than the full-length protein was used for two reasons. First, t-UL9 causes a much simpler pattern in gel shift experiments than UL9, probably because it does not have a tendency to aggregate. Second, t-UL9 has been reported to be a monomer in solution, a fact that simplifies the interpretation of binding stoichiometry measurements. A recombinant baculovirus was constructed which expresses a polypeptide comprising amino acids 534-851 of UL9, with an oligo-histidine affinity tag placed at the amino terminus to facilitate the isolation of chemically homogeneous protein of high specific activity. The protein was labeled in vivo with [H]leucine and was purified to chemical and radiochemical homogeneity in a two-step process from the infected nuclear extract using nickel-agarose affinity chromatography and gel permeation chromatography. The silver-stained polyacrylamide gel of the peak fractions from the gel permeation column and the corresponding fluorogram of the silver-stained gel are shown in Fig. 1. The minor band migrating at a slightly lower molecular weight from t-UL9 was seen in varying proportions to the major band in different t-UL9 preparations and is likely to be a proteolytic product of t-UL9. The hydrodynamic properties(22) , DNase I footprint pattern(2, 22) , and gel shift pattern(1, 2, 7, 15, 16, 23) of the H-labeled His-tagged t-UL9 were the same as those described previously for similar untagged molecules. The molar extinction coefficient of His-tagged t-UL9 was calculated to be 37,830 M cm, using the method of Gill and von Hippel(28) . The absorbance at 280 nm of t-UL9 denatured with 6 M guanidinium HCl, however, was 10% lower than the absorbance of the native protein. The molar extinction coefficient of the H-labeled t-UL9 was therefore adjusted to 34,047 M cm, and the specific activity was calculated to be 27,978 dpm/pmol.

Figure 1: Silver stain and fluorogram of polyacrylamide gel containing the peak fractions of the final step of purification of H-labeled t-UL9. One µl of each of the load and peak fractions from the Smart/Superose 12 column was run on a 4-20% polyacrylamide gel(27) . The gel was stained with silver, soaked in Amplify, dried, and subjected to autoradiography. The fraction numbers are listed across the top of the gel. For column calibration, Bio-Rad molecular mass standards -globulin (160 kDa), ovalbumin (44 kDa), and ribonuclease A (17 kDa) eluted in fractions 15, 22, and 28, respectively.

Varying amounts of H-labeled t-UL9 were mixed with a fixed amount of P-labeled 24-base pair double-stranded oligonucleotide containing site I. The protein-DNA complexes were separated from the unbound DNA by nondenaturing polyacrylamide gel electrophoresis. As seen in Fig. 2, a single lower mobility band was seen at all t-UL9 concentrations. The lower mobility complex in each lane was excised from the gel, dissolved, and the amount of H and P radioactivity determined by liquid scintillation counting. The results from this experiment are shown (Fig. 2). The labeled bands contained 1 10 to 1 10 dpm of H and P radioactivity, with H dpm in approximately 3-fold excess over P dpm; both amounts are in a range where counting errors are negligible. In a separate experiment similar to that shown in Fig. 2, one lane of the gel was cut into 0.5-cm slices, and the radioactivity in each slice was determined. As depicted graphically in Fig. 3, the H-labeled protein was associated predominately with the P-labeled DNA and was not distributed throughout the gel. The ratio of t-UL9 monomers to UL9 binding sites was determined to be 2.0 ± 0.1 (n = 13).

Figure 2: Autoradiogram of a double-label gel shift experiment using H-labeled t-UL9 and P-labeled site I oligonucleotide. H-Labeled t-UL9 (27,978 dpm/pmol) was titrated into reaction mixtures containing 5 pmol of 32P-labeled site I oligonucleotide (19,111 dpm/pmol): lane 1, no added protein; lanes 2-6, 3, 6, 9, 12, and 18 pmol of H-labeled t-UL9, respectively. The protein-DNA complexes were separated from the unbound DNA fragments on a 5% nondenaturing polyacrylamide gel. The gel was exposed to x-ray film at room temperature while wet, the lower mobility bands (at the position indicated by the arrow) were cut from the gel, and the slices were dissolved and counted. The H dpm and P dpm of each excised gel slice are listed below the corresponding lower mobility band.

Figure 3: Plot of radioactivity versus distance migrated. A representative lane from an experiment similar to that shown in Fig. 2was sectioned into 0.5 1.0-cm slices from the well to the bottom of the gel. The slices were dissolved, counted, and the data plotted. The slower mobility peak in the plot corresponds to the position on the autoradiogram of the gel of the low mobility complex. The faster mobility peak in the plot corresponds to the position on the autoradiogram of the gel of the faster mobility complex.

To control for possible errors that might have been introduced by the use of a synthetic oligonucleotide, a DNA substrate prepared by a different process was also used. HSV-2 ori was excised from highly purified plasmid DNA, endlabeled with P, and the labeled DNA fragment was purified by agarose gel electrophoresis. The gel shift assay was performed as described above with the synthetic oligonucleotide. The addition of H-labeled t-UL9 to reactions containing this DNA fragment resulted in the appearance of two lower mobility complexes, with the faster mobility of the two predominating at low t-UL9 concentrations and the slower mobility complex predominating at higher concentrations of t-UL9 (Fig. 4). DNase I footprint analysis of the DNA in the two complexes revealed protection over sites I and II only, with more complete protection of both sites I and II in the lower mobility complex (data not shown). The ratio of monomer t-UL9 molecules to UL9 binding sites was 1.8 ± 0.1 (n = 15) for the higher mobility complex and 3.7 ± 0.4 (n = 15) for the lower mobility complex. These data are in close agreement with those obtained with the synthetic oligonucleotide that contained a single UL9 binding site. Thus, two UL9 binding domains are necessary and sufficient for binding to a single UL9 binding site. These findings, therefore, confirm the previously published results of Stabell and Olivo(2) . In addition, these results show that four UL9 binding domains are bound to ori. Taken together, these data are consistent with a model (Fig. 5) in which two UL9 dimers are bound to ori, one each at site I and site II.

Figure 4: Autoradiogram of a double-label gel shift experiment using H-labeled t-UL9 and P-labeled ori. H-Labeled t-UL9 was titrated into reaction mixtures containing 0.6 pmol of 32P-labeled ori: lane 1, no added protein; lanes 2-6, 0.8, 1.7, 3.3, and 6.6 pmol of H-labeled t-UL9, respectively. The protein-DNA complexes were separated from the unbound DNA fragments on a 5% nondenaturing polyacrylamide gel. The gel was exposed to x-ray film at room temperature while wet, the bands were cut from the gel, and the slices were dissolved and counted. The ratio (mean of all experiments, n = 15) of H-labeled t-UL9 molecules to P-labeled ori molecules is reported next to the position of the corresponding band.

Figure 5: Model diagram depicting the binding of ori by: A, t-UL9; and B, UL9. A/T is the A/T-rich region between the two UL9 binding sites in ori. The bipartite shaded regions of sites I and II depict the overlapping inverted binding half-sites. The small, lightly shaded area between monomer UL9 molecules of the UL9 homodimer represents the monomer-monomer interaction between UL9 molecules of the homodimer molecule. The larger shaded regions between UL9 homodimers bound to ori represent the interaction between UL9 homodimers as suggested by the cooperative binding of UL9 to ori.

It is clear that these conclusions are critically dependent on the accuracy of the measurements. Since the standard error derived from the average of 15 independent measurements was between 1 and 3%, it seems unlikely that the conclusions are affected by random experimental error. Possible sources of systematic error, and the potential magnitude of such errors, are, however, worth noting. In addition to the quantity of H and P radioactivity present in various samples, the only other measured values in the described experiments were the chemical amounts of DNA and protein. In both cases, these quantities were determined spectrophotometrically, and therefore the conclusions depend on the accuracy of the extinction coefficients used. The extinction coefficient of double-stranded DNA at 260 nm is known precisely and is essentially independent of the sequence of the DNA(3) . In addition, as an internal control in our determinations of the binding stoichiometry, two different DNA molecules isolated from independent sources were used, and essentially the same result was obtained with each. The specific activity of the P-labeled DNA, therefore, is unlikely to be a source of significant error in the calculation of the binding stoichiometry.

Precise determination of the extinction coefficient of t-UL9 is somewhat more problematic. In contrast to DNA, proteins do not have a uniform extinction coefficient at 280 nm. The method of Gill and von Hippel (28) was used to derive the extinction coefficient for t-UL9. This method is based on the demonstration by Edelhoch (40) that only tryptophan, tyrosine, and cysteine residues contribute significantly to the measured optical density of a denatured protein at 280 nm. The extinction coefficient of the protein of interest is calculated by multiplying the number of residues of each of the three absorbing amino acids by the molar extinction coefficients of model peptides containing these residues. This method for calculating molar extinction coefficients was calibrated by calculating the extinction coefficients for 18 globular proteins whose extinction coefficients were accurately known(28) . The mean deviation between the experimental and the calculated values was +3.8% ± 6%, with no deviation greater than +14.9% (for human serum albumin). Since the method was calibrated using native proteins, the largest potential error in this calculation is likely to be the effect of tertiary structure of the native protein on the absorbance of the individual absorbing amino acids. This effect was measured by comparing the absorbance at 280 nm of native and denatured t-UL9, as described under ``Experimental Procedures.'' A small difference (10%) between the absorbance of the native and denatured proteins was observed, and the extinction coefficient of t-UL9 was adjusted appropriately. On the basis of this small difference in absorbance between the native and denatured protein and on the basis of the prior results of Gill and von Hippel, the estimated error in the calculated value for the extinction coefficient of t-UL9 is less than 15% and is likely to be much lower.

Recently, two other laboratories have used gel shift assays to measure the binding stoichiometry between t-UL9 and the origin; the results of these two studies were not in agreement. Stabell and Olivo (2) titrated Fab fragment directed at the carboxyl terminus of UL9 into a gel shift mixture containing t-UL9 bound to a single UL9 binding site and detected two bands of lower electrophoretic mobility, suggesting that two t-UL9 molecules bound the site. Martin et al.(1) used in vitro transcription/translation to obtain t-UL9 and a slightly truncated t-UL9 molecule. In gel shift experiments in which both of these forms of t-UL9 were present, the investigators observed only two complexes, the mobilities of which corresponded to the mobilities of the complexes observed with each form of t-UL9 alone. This result suggested that no heterodimers between the larger and smaller polypeptides were formed during cotranslation and therefore that t-UL9 bound to a single binding site as a monomer. The disparate conclusions from these sets of experiments are difficult to reconcile; both, however, required significant assumptions about the gel shift banding pattern without knowledge of the composition of the bands. The experiments presented in this paper have significant advantages over the indirect approaches described above. Foremost, no assumptions are required as to the interpretation of the banding pattern in the gel shift experiments, as the composition of each complex was determined directly. In addition, the t-UL9 used in the double-label gel shift experiments was highly purified and well characterized, and secondary heterologous protein binding was not required for the stoichiometric determinations. The conclusions, therefore, are based on direct measurements of physical constants and require only that the labeled protein and DNA were chemically pure and that the extinction coefficient for t-UL9 was determined accurately. The results, then, confirm those of Stabell and Olivo(2) , that t-UL9 binds to a single high affinity UL9 binding site as a dimer, and they extend the findings to demonstrate that two t-UL9 dimers bound to ori.

Taken together, the simplest interpretation of these data is that one full-length UL9 dimer binds to a single UL9 binding site and that two full-length UL9 dimers bind to ori (Fig. 5). The underlying assumption of this model is that the results obtained with t-UL9 can be extrapolated directly to what occurs with the full-length protein. Other more complicated models, however, are consistent with the available data. For example, it is possible that full-length UL9 monomers comprising a UL9 dimer are oriented so that the two monomeric units cannot both bind to the same DNA binding site. According to this model, a single binding site would be occupied by two UL9 dimers, with two unoccupied monomeric units available to bind to another site. To address this question, the double-label gel shift experiment was employed using -H-labeled full-length UL9. As has been reported previously(23, 38) , UL9 and the site I oligonucleotide formed a large number of complexes that were resolvable by gel electrophoresis. Moreover, the complexes did not differ in the protein:DNA ratio by an integral number of UL9 molecules, nor was there a monotonic increase or decrease in the protein:DNA ratio of the complexes as a function of electrophoretic mobility (data not shown). We currently have no simple interpretation of these data, although we assume that the large number of protein-DNA complexes formed with full-length UL9 is related to its tendency to aggregate. In any case, these data do not distinguish between the model presented in Fig. 5and alternative models.

The binding of two t-UL9 monomers to a specific binding site is highly cooperative, since no intermediate t-UL9-site I complex containing a single monomer of t-UL9 was detected (see Fig. 2and Fig. 4). The simplest explanation for this high degree of cooperativity is that t-UL9 monomers interact with each other. As noted above, t-UL9 is a monomer in solution, at least at concentrations less than 1 10M. Higher order association between t-UL9 molecules in concentrated solutions has been observed, however, ()()and experiments designed to confirm this finding using highly purified protein are in progress. There are also other possible explanations for the highly cooperative nature of t-UL9 binding to site I. For example, it has been reported that UL9 binding to the origin distorts the DNA near its binding site(39) . It is possible that the change in free energy associated with this DNA conformational change contributes to cooperativity. Alternatively, the t-UL9 may undergo conformational changes upon binding to DNA that increase the magnitude of protein-protein interactions. A rigorous analysis of the energetics of UL9-DNA interactions will be required to distinguish among these possibilities.

The finding that two UL9 DNA binding domains bind to a single binding site predicts that the DNA sequence of the binding site should contain a 2-fold axis of symmetry. The consensus 10-base pair UL9 recognition site 5`-CGTTCGCACT, however, does not contain a simple 2-fold axis of symmetry. Koff and Tegtmeyer (12) have suggested that similar to the SV40 T antigen recognition sequence, site I is composed of inverted overlapping repeats that could be the binding half-sites for UL9. A more recent mutational analysis of ori by Hazuda et al.(9) has provided data consistent with this hypothesis. Although it would be without precedent, it is also possible that the UL9 homodimer binds asymmetrically to DNA. Further, more detailed investigation of the binding of UL9 to its recognition sequence will be required to determine the exact nature of this binding interaction.

FOOTNOTES

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Laboratory of Viral Diseases, NIAID, NIH, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-8274; Fax: 301-402-2622.

()

The abbreviation used is: HSV, herpes simplex virus.

()

S.-j. Um and R. McMacken, personal communication.

()

E. C. Stabell and P. D. Olivo, personal communication.

()

D. S. Fierer and M. D. Challberg, unpublished results.

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