Structural Elements Required for the Cooperative Binding of the ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 24, Issue of August 25, pp. 17424-17429,1992 Printed in V.S.A.

Structural Elements Requiredfor the Cooperative Binding of the Herpes Simplex Virus Origin Binding Protein to oris Reside in the N-Terminal Part of the Protein* (Received for publication, April 3, 1992)

Per EliasSQ, Claes M. GustafssonS, Ola Hammarsten$, andNigel D. Stowll From the $Department of Medical Biochemistry, University of Goteborg, Medicinaregatan 9, S-413 90 Goteborg, Sweden and the lIMediea1Research Council Virology Unit, Institute of Virology, Church Street, Ghgow GI I 5JR, United Kingdom

The origin binding protein (OBP)of herpes simplex indicated by profound alterations of the DNase I digestion virus type 1 is required to activate a viral origin of pattern of the entire oris region (Elias et al., 1990). An replication in vivo. We have used intact OBP as well electron microscopy study has recently demonstrated that as a truncated formof the protein expressed in Esche- these complexes are exclusively located at oris sequences richia coli to investigate the protein-protein interac- encompassing approximately 120 base pairs of DNA (Rabkin tions, as well as the protein-DNA interactions involvedand Hanlon, 1991). A noteworthy feature of the OBP-oris in the formation of a nucleoprotein complex at a viralcomplex is a structural alteration of the 18-nucleotide-long origin of replication (oris) in vitro. The salient findings demonstrate that the N-terminal part of OBP is AT-rich spacer region in oris induced by OBP (Koff et al., required for the cooperative binding of OBP to three 1991). What are the structuralfeatures of OBP that are required sites (boxes I, 11, and 111) within oris. A detailed model for the interaction of OBP with the viral origins of for the formation of the nucleoprotein complex at oris?Here, we use nativeOBP, as well as a truncated form of OBP replication oris and oriLis presented. (AOBP) consisting of the DNA-binding domain to demonstrate that cooperative interactions mediated by the N-terminal part of the protein are involved in the binding of OBP Considerable effort has been made in thesearch for factors to threesites: boxes I, 11, and 111. A tentative structuralmodel and conditions that govern the initiation of DNA replication for the OBP-oris complex is discussed on the basis of these at chromosomal origins of replication. In some cases the observations. successful development of in uitro systems that faithfully EXPERIMENTAL PROCEDURES execute origin-dependent DNA synthesis has allowed the Plusmids and Oligonucleotides-Oligonucleotides synthesized in identification of a number of crucial steps that,despite large structural variations between species, seem to form a unifying the trityl-on mode on a Beckman DNA SM synthesizer were purified on OPC cartridges from Applied Biosystems and used to create wild scheme for the activation of an origin of replication (Kornberg type and mutant versions of oris, essentially as previously described and Baker, 1992). Foremost among these steps is the forma- (Elias et al., 1990). The resulting fragments were inserted at the tion of a highly organized nucleoprotein complex at theorigin BarnHI site in the polylinker of pTZ18r. Plasmid DNA was purified of replication (Fuller et al., 1984). This event leads to a local by standard techniques and sequenced. In the investigations described unwinding of duplex DNA where the assembly of replication below, three plasmids with different versions of the HSV-1 oris forks can take place (Bramhill and Kornberg, 1988; Dean et sequence wereused. The plasmid pCG 5, a wild type construct, nucleotides 1-75 from Fig. 1 with the addition of BamHIal., 1987; Schnos et al., 1988). The prime organizers of these contained compatible ends. The plasmid pCG 11 also contained nucleotides 1complexes are sequence-specific DNA-binding proteins that 75 from Fig. 1 with the only alteration being that two T to G frequently have ATPase or helicase activities (Stahl et al., transversions had been introduced at positions 24 and 25.pCG 11 1986; Sekimizu et al., 1987). therefore contained the sequence CGGGCGCACTT in boxI. As We have focused our interest on one of these proteins, the described for pCG 5, BamHI-compatible ends were added. Finally, origin binding protein (OBP)’ of herpes simplex virus type I the plasmid PORI lb, a wild type construct, contained the entire oris described in Fig. 1. (Elias et al., 1986; Elias and Lehman, 1988; Olivioet al., 1988; sequence Cloning and Expression of AOBP-The UL 9 gene from the HSVWeir et al., 1989).This protein bindsspecifically to two largely 1 KOS strain was cloned into the overexpression vector PET-3a as homologous viral origins of replication, oriL and oris(Weller previously described (Elias et al., 1990). A deletion mutant between et al., 1985; Stow and McMonagle, 1983). It is also an ATPase two BamHI sites at positions corresponding to amino acids 10 and and DNA helicase (Bruckner et al., 1991). The formation of 535 was made. Expression of AOBP was obtained in Escherichia coli a large nucleoprotein complex between OBPandoris is BL 21 (DE 3) pLysS cells upon induction with 1 mM isopropyl-l*This research was supported by the Swedish Cancer Society Grant 2552-B89-02XB. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 To whom correspondence should be addressed. Tel.: 46-31-8534-86; Fax: 46-31-41-61-08. The abbreviations used are: OBP, origin binding protein; HSV-1, herpes simplex virus type 1; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; MES, 4-morpholineethanesulfonic acid.

thio-P-D-galactopyranosideas previously described (Elias etal., 1990). Purification of OBP and AOBP-The DNA binding activities of OBP and AOBP were measured during the course of purification by a nitrocellulose filter-binding assay (Elias et al., 1990). One unit of OBP retains 1 fmol of a box I duplex oligonucleotide on the filter (Elias and Lehman, 1988). The purification of OBP followed the previously described protocol with the modifications described below. A more efficient and reproducible extraction of OBP was obtained by suspending the washed bacteria from two 2-liter cultures in 320 ml of 50 mM Na+-HEPES, pH 7.6, 1 mM DTT, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 2 mM benzamidine. Nonidet P-40, 32mlof 10% v/v,

17424

Protein-Protein Interactions Origin an at was added, and the suspension was incubated at 25 "C for 30 min. T h e clear viscous lysate was diluted with 1 volume of 50 mM Na+HEPES, pH 7.6, 0.2 M NaC1, 20% v/v glycerol, 1 mM DTT, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 2 mM benzamidine. The viscous lysate was sheared using the Ultraturrax homogenizer at high speed, centrifuged, diluted, and applied on a S-Sepharose column exactly as previously described (Elias et al., 1990). A final chromatography step was added to the protocol using a Mono S column eluted with a gradient of 0.1 M NaCl uersus 1 M NaCl in 20 mM Na+-HEPES, pH 7.6, 10% glycerol, 0.1% Nonidet P-40, and 1 mM DTT. Dialysis and storage of OBP was as before (Elias et al., 1990). AOBP could not be purified following the protocol described above, and a novel procedure was therefore developed. Two 2-liter cultures were grown and induced as for the expression for OBP. The cells were harvested and washed as previously described (Elias etal., 1990). The bacterial pellet was suspended in 150 ml of 50 mM Na+-HEPES, p H 7.6, 10% glycerol, 2 mM EDTA, 10 mM NaHS03, 1 mM DTT, 2 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride (buffer A), using an Ultraturrax homogenizer at low speed. The cells were lysed by the addition of 15 ml of 10% Nonidet P-40 and kept at 25 "C for 30 min. The clear suspension was transferred to ice and 150 ml of ice-cold 0.2 M NaCl in buffer A was added. The viscous lysate was sheared by a few brief pulses using the Ultraturrax homogenizer at high speed and subsequently centrifuged a t 4 "C for 15 min at 15,000 rpm in the Beckman J A 20 rotor. The pH was adjusted to 6.3 by the addition of 1 M Na+-MES, pH6.0. The supernatant was diluted with 2 volumes of 50 mM NaCl, 20 mM Na+-MES, pH6.0,10% glycerol, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine (buffer B). This solution was chromatographed on a 400-ml column of DEAE-Sepharose CL-GB (Pharmacia LKB Biotechnology Inc.) equilibrated with buffer B and developed with a linear gradientof 1.5 liters of buffer B uersus 1.5 liters of 1 M NaCl in buffer B. Further purification was achieved on a 20-ml column of calf thymus DNA coupled to Sepharose CL-2B as described (Arndt-Jovin etal., 1975). The column was equilibrated with 50 mM NaCl, 20 mM Na+-HEPES, p H 7.6, 1 mM DTT, and 10% glycerol (buffer C). After loading, the column was washed with buffer C. AOBP was eluted with 0.25 mM NaCl in buffer C. The final step of purification used a sequencespecific DNA affinity column previously described for OBP (Elias and Lehman, 1988). The active fractions were loaded on the affinity column equilibrated with 0.3 M NaCl in buffer C. AOBP was eluted with 1 M NaCl in buffer C. The active fractions were concentrated using Centriprep 10 (Amicon) and dialyzed against 0.2 M NaC1,20 mM Na+-HEPES, pH 7.6,l mM DTT, and50% glycerol. This fraction could now bestored at -20 'C for several months without a significant loss of activity. Electrophoresis and Gel Filtration of AOBP-SDS-polyacrylamide gel electrophoresis of AOBP was performed on 15% gels using the LMW and HMW standards from Bio-Rad as markers (Laemmli, 1970). The Stokes radius was determined in gel filtration experiments on Sephacryl S 200 HR (Pharmacia LKB Biotechnology Inc.) in 0.5 M NaC1,50 mM Na+-HEPES, pH 7.5, 1 mM DTT, and 10% glycerol. Lysozyme (14,400), ovalbumin (45,000), aldolase (160,000) were used a s markers. K., was calculated from the equation K., = V, - V,/V, VO. DNase Z "Footprinting"-Restriction fragments containing orisderived sequences from the plasmids pCG 5, pCG 11, and PORI l b were end-labeled with [cv~'P]~TTP(3000 Ci/mmol) at the Hind111 or EcoRI sites of the polylinker sequence using DNA polymerase I large fragment. The reaction mixture contained in a total volume of 20 p1 of 0.1 M NaCl, 50 mM Na+-HEPES, pH 7.5, 5 mM MgC12, 0.1 mM EDTA, 0.5 mM DTT, 10% glycerol, and 0.25 nM (0.5 ng) 32Plabeled restriction fragment (30,000 cpm). A 20-fold excess of OBP or AOBP (100 units as determined in the filter-binding assay) was included as indicated. In the competition experiments, calf thymus DNA (40 and 400 ng, respectively) was added prior to the addition of protein. Incubation was for 10 minon ice followed by 10 min at 20 "C. 2 pl of DNase I (20 pg/ml) was added, and theincubation was allowed t o proceed for 2 min. The incubation was stopped by the addition of 6 pl of 0.5 M EDTA, 0.1 mg/ml calf thymus DNA and transferred to ice. Following a dilution with 75 p1 of TE, thesamples were extracted with phenol/chloroform for 10 min. DNA was recovered by ethanol precipitation and dissolved in 3 1 1 of water and 2 pl of formamide dye solution. The denatured samples were analyzed on 8% sequencing gels run at 55 'C a t 2500 V. Autoradiography using Fuji RX x-ray film was for 3 days at room temperature without intensifying screen.

of Replication

17425

The autoradiographs were scanned using the Molecular Dynamics computing densitometer 300 A. Line graphs were obtained using the wide line integration option. The graphs presented in this paper were produced using Sigma plot version 4.0 (Jandel Scientific). RESULTS

Properties of the DNA-binding Domain OBP-It of has been demonstrated that a fusion protein containingthe C-terminal 316 amino acids of OBP can bind specifically to the binding sites box I and I1 within oris (Weir et al. (1989); see also Deb and Deb (1991)). This fusion protein forms only one type of complex with a duplex oligonucleotide containing the box I sequence when examined in gel retardation experiments (Weir et al., 1989). On the other hand, OBP of full length forms several discrete complexes when analyzed in a similar way (Elias and Lehman, 1988). In order to investigate the possibility that protein-protein interactions between the N-terminal parts of OBP monomers are responsible for the cooperative binding of OBP to oris, we have now made a deletion mutant of OBP (AOBP) consistingof amino acids 1-10 fused to 535-851 (Fig. 1).This construct was cloned into the T7based expression vector pET-3A, and expression of AOBP was achieved in E. coli BL21 (DE 3)pLys S cells (Rosenberg et al., 1987). AOBP was purified to near homogeneity using sequence-specific affinity chromatography. The purified protein had a molecular weight of 36,000 as determined by SDSpolyacrylamide gel electrophoresis and existed as a monomer in solution when examined in gel filtration experiments (Fig. 2). On the other hand, native OBP was isolated as a dimer (results not shown; see also Bruckner et al. (1991)). AOBP was found to form a complex with duplex oligonucleotides with the same specificity as full-length OBP, as determined by competition experiments with mutant duplex oligonucleotides using the nitrocellulose filter-binding technique (results not shown). The stability of a complex between AOBP and a 26-base pair duplex oligonucleotide containing the box I sequence was similar to that formed by intact OBP (Fig. 3). These observations agree with the results recently presented by others (Hazuda et al., 1991) and suggest that all structural elements required for sequence-specific binding to DNA are contained within the C-terminal316 amino acids of OBP. OBP Binds Specifically to Three Sites within oris-OBP has previously been shown to bind to two sites within oris, boxes I and I1 (Elias and Lehman, 1988; Elias et al., 1990). There was a 5-10-fold difference in affinityfor these two sites when analyzed by filter-binding experiments using radiolabeled duplex oligonucleotides as substrates (Elias and Lehman, 1988). OBP did notbind to duplex oligonucleotides containing the putative third binding site, box I11 (Elias et al., 1990; Weir and Stow, 1990).A DNase I-footprinting study, however, displayed an altered digestion pattern also in the box I11 region (Elias et al., 1990). Here, we have used the oris-containingrestriction fragment from the plasmid PORI l b in a DNase I-footprintinganalysis to demonstrate that specific protection was indeed obtained for box I11 (Fig. 4). This effect has been highlighted in densitometric scans at high resolution (Fig. 5). Furthermore, the patternof protection was not altered by the addition of a 100-1000-fold excess of competing calf thymus DNA (Figs. 4 and 5). Itis also noteworthy that the truncatedform of OBP, AOBP, did not interact with box I11 (Figs. 4 and 5). On the basis of the observation mentioned above that OBP bound a 27-base pair duplex oligonucleotide containing box I11 very poorly, we conclude that the intrinsically very weak interaction between OBP and box I11 was greatly stabilized within the nucleoprotein complex at oris. Using full-length OBP, we observed an altered digestion

Protein-Protein Interactions at

17426

a n Origin of Replication

A. oriL

-m

oris B.

Box I11

Box I

5’ GATCCCGGGTRRAAGAkGT~GA&CGCGA&GCGTTCGCA~TTCGTCCCA& 1

10

40 20

30

Box I1 TATATATATATTATTAGGGGGAAGTGCGA-mCTGGCGCCETGCCCGATC 50 60 10 80

Molecularweight

3’

C. 1

MPFVGGAESG DPLGAGRPIG DDECEQYTSS VSLARMLYGG DLAEWVPRVH

51

PKTTIERQQH GPYTFPNASR PTARCVTVVR APKSGKTTAL IRWLREAIH

101 SPDTSVLVVS CRRSFTQTLA TRFAESGLVD FVTYFSSTNY IMNDRPFHRL 151

0.8

1

-1

> Y

IVQVESLHRV GPNLLNNYDV LVLDEVMSTL GQLYSPTMQQ LGRVDALMLR

201 LLRICPRIIA MDATANAQLV DFLCGLRGEK NVHWVGEYA MPGFSARRCL 251 FLPRLGTELL QAALRPPGPP SGPSPDASPE ARGRTFFGEL EARLGGGDNI 301 CIFSSTVSFA EIVRRFCRQF TDRVLLLHSL TPLGDVTTWG QYRVVIYTTV

351

VTVGLSFDPL HFDGMFRYYK PMNYGPDMVS VYQSLGRVRT LRKGELLIYM

401

DGSGARSEPV FTPMLLNHVV SSCGQWPAQF SQVTNLLCRR FKGRCDASAC

451 DTSLGRGSRI YNKFRYKHYF ERCTLACLSD SLNILHMLLT LNCIRYRFWG 501 HDDTLTPKDF CLFLRGVHFD ALRRQRDLRE LRCRDPERSL PAQAAETEEV 551 GLFVEKYLRS DVAPAEIVAL MPNLNSLMGR TRFIYLALLE ACLRVPMRTR 601

SSAIFRRIYD HYATGVIPTI NVTGELELVR LPPTLNVTPV WELLCLCSTM

651

AARLHWDSAA GGSGRTFGPD DVLDLLTPHY DRYMQLVFEL GHCNVTDGLL

701 LSEEAVKRVA DALSGCPPRG SVSETDHAVA LFKIIWGELF GVOMRKSTQT 751

FPGAGRVKNL TKQTIVGLLD AHHIDHSACR THRQLYALLM AHKREFAGAR

Molecularweight

FIG.2. AOBP is a monomer in solution. A , the molecular weight of 36,000 for AOBP (filled circle)was estimated by gel electrophoresis on15% SDS-polyacrylamidegels. The molecular mass markers (opencircles) were 31, 45, 66, 97, 116, and 200 kDa. B, gelfiltration of AOBP (filled circle) on Sephacryl S 200 HR. Markers (open circles) were lysozyme (14,0001, ovalbumin (45,000), and aldolase (160,000). K., was calculated as described under “Experimental Procedures.”

801 FKLRVPAWGR CLRTHSSSAN PNADIILEAA LSELPTEAWP MMQGAVNFST 851

L

FIG.1. A presentation of the viral origins of replication oris and oriL, the sequences of the minimal HSV-1origin of replication, oris, and the origin-binding protein, OBP. A , a comparison of oris and oriL. The long palindrome of oriL contains four repeats of the recognition sequence for OBP (open areas). The three binding sites for OBP in oris arereferred to asboxes I, 11, and 111 in the text. B, the sequence of HSV-1 oris presentin the plasmid PORI lb, a wild type construct. Nucleotides that have been shownby filter-binding experimentsto be required for sequence-specific binding of OBP to oligonucleotides are shown in bold. C, the amino acid sequence of OBP, the product of the UL 9 gene (McGeoch et al., 1988). The putative ATP-binding site been has highlighted inboldface (Hodgman, 1988a, 1988b). AOBP consists of amino acids 1-10 fused t o 536-851 (underlined). pattern of the AT-rich spacer sequence corresponding to nucleotides 39-56of Fig. 1. Furthermore, a novel enhanced cut of the phosphodiester bond between A and C of the recognition sequence CGTTCGCACTT was obtained (Figs. 4 and 6). These features were completely absent when AOBP was used in the footprinting analysis (Figs. 4 and 6). The conformational changes of oris induced by OBP must therefore depend onprotein-protein contacts involving the Nterminal partof OBP. Cooperative Binding to orisIs Mediated by the N-terminal Part of OBP-We have previously noted that OBPcould bind with high affinity to a mutated box I when this sequence was present in the context of oris but notwhen it appeared in an isolated 26-base pair duplex oligonucleotide, thus indicating cooperative binding of OBP to oris (Elias et al., 1990). We have now extended these observations using the plasmids pCG 5, a wild type construct, and pCG 11, where the box I sequence was altered to read CGGGCGCACTT as described under “Experimental Procedures” (see also Elias et al., 1990;

L L

0

600

1200

Time(seconds)

FIG.3. The stability of complexes formed by OBP (filled with a duplex oligonucleotide circles) and AOBP (open circles) containing box I (oligonucleotide 17/18in Elias and Lehman (1988)).The measurements were performed on ice using the nitrocellulose filter-binding technique. A preformed OBP-oligonucleotide complex in 15 pl was challenged with a 100-fold excess of unlabeled oligonucleotide in a total volume of 1 ml a t time 0. The diluted samples were filtered at the times indicated. The value 100 denotes the amountof DNA retained on thefilter a t time 0.

Hernandez et al., 1991). In a DNase I-footprinting experiment where OBP or, alternatively, AOBP was added to restriction fragments containing oris from the plasmids pCG 5 (wild type) and pCG 11 (box I mutation), we could now observe that only full-length OBP bound to the mutated version of box I (Figs. 6 and 7). In addition, protection against DNase I cutting of the box I11 region of restriction fragments derived from pCG 5, as well as pCG 11 was observed only in the presence of intact OBP (Fig. 6 and 7). Again, the DNase I digestion patterns did not change upon the addition of competing calf thymus DNA, lending support to the interpretation that a stable nucleoprotein complex was formed (results not shown). These findings clearly demonstrate that the binding of OBP to boxes I, 11, and I11 is sequence-specific and highly

Protein-Protein Interactions at Plasmid Strand Additions AOBP OBP CT-DNA lOOx

" U "

- + -

"+

--

-

PORI u u "

++ + -

an Origin of Replication

( rt )

1 1 1

1 1

- + -

"

"+

-

- -

17427 Box

1

$0.

I

I

++ +-

b

60

fio

Ill

30

Box I1 70

20

80

10 4

1

1

"

"-

~

-_

"

FIG. 4. OBP binds to three sites within oris. A DNase Ifootprinting analysis of the nucleoprotein complexes formed between an oris-containing restriction fragment from the plasmid pORIlb, a wild type construct described under "Experimental Procedures," and a 20-foldexcess of AOBP or full-length OBP, respectively. The numbers refer to the nucleotide sequence in Fig. 1. The numbers corresponding to theupper strand arepresented at theleft, and those for the lower strand are at theright. Boxes III, I, and II contain the nucleotides 6-16, 22-32, and 62-72. The AT-rich spacer sequence is found between nucleotides 39 and 56. The arrowheads indicate the position of the unprotected A-C bond within the recognition sequence CGTTCGCACTT.

cooperative. Since OBP but notAOBP binds cooperatively to oris, we conclude that essential structural information for these cooperative protein-protein interactions reside in the N-terminal partof OBP. DISCUSSION

We have in this paper described an investigation of some of the protein-protein interactions and DNA-protein interactions thatoccur within anucleoprotein complex at anorigin of replication, oris, of herpes simplex virus type 1. In this context, we have previously noted that two binding sites within oris, box I and box 11, have an intrinsically high affinity for the origin-binding protein (Elias et al., 1990). We have now observed that thesetwo sites arealso recognized by AOBP, a truncated version of OBP in which 523 N-terminal amino acids have been deleted. A third binding site within oris, box 111, has a low intrinsic affinity for OBP due to an altered recognition sequence but can nevertheless be recog-

FIG. 5. Cooperative binding of OBP to boxes I and I11 of oris. The scans were performed on the autoradiograph shown in Fig. 4. The dotted line in all panels correspond to thescan of panel a. The solid lines correspond to experiments where OBP or AOBP were used. a, no protein added (6th lane); b, AOBP added (7th lane), c, OBP OBP and 40 ng of calf thymus DNA added (9th added (8th lane); d, lane); e,OBP and 400 ng of calf thymus DNA added (10th lane).

nized by OBP within an OBP-oriscomplex. AOBP, however, is ineffective in this respect. The cooperative protein-protein interactions occurring within the nucleoprotein complex at oris were also demonstrated when the binding of OBP to an oris construct with a mutated box I sequence was monitored. Here, we observed no binding to amutated box Iinthe presence of AOBP, whereas intact OBP still recognized this site with high affinity. Apparently, the structuralinformation for cooperative binding of OBP to oris is contained within the N-terminal part of the protein. We have previously described some of the features that need to be accommodated within a model of an OBP-oris complex (Elias et al., 1990). We now present the essential features of this model more explicitly in Fig. 8. The basic assumption is that OBP forms a rigid protein corewhen bound to oris and that the same structure is formed when OBP is bound to oriL. In its simplest version, the model requires two OBP dimers. The C-terminal domain of each OBP monomer binds one recognition sequence. There are three repeats of this sequence in oris, boxes I, 11, and 111, and studies with mutant origins imply that all of these sites do play a role in the initiation of DNA synthesis (Hernandez et al., 1991; Weir and Stow, 1990). If we assume a complex with two dimers, one of the four DNA-binding domains will therefore be unoccupied or only loosely attached to DNA. In the case of oriL, all four DNA-binding domains will be occupied. The protein-protein interactions responsible for the cooperative binding to boxes I and I1 involve the N-terminal part of the protein. The nature of the protein-protein contacts required for the binding of OBP tobox I11 is less clear. In this instance, the N-terminal parts may participate directly with

Protein-Protein Interactions at an Origin of Replication

17428 Plasmid

DCG 5

Strand Additions AOBP OBP

1 1 11 1 1

(wt)

- + " +

DCG 11 (box1 mut)

DCG 5 (wt)

DCG 11 (box1 nut)

- + -

"*

kyw Box l

Box I

70

Box 11 60

50

"- ---

III

r "

40

BOX

30

I

20

BOX I11

10

1

~.

FIG.6. Cooperative binding of OBP to oris ismediated by the N-terminal part of OBP. A DNase I footprint examining the

FIG.8. A model of the interaction between OBP and the viral originsof replication of oriL and oris. The model is based

interaction between full-length OBP or AOBP with the oris-containing restriction fragments from the plasmids pCG 5 (wild type ( w t ) ) and pCG 11 (bowl mut) (see "Experimental Procedures"). The numbers refer to the sequence in Fig. 1. The numbers corresponding to the lower strand are presented at the left, and those for the upper right. strand are at the

on the simple assumption that each recognition sequence is bound by a single DNA-binding domain of OBP. Two OBP dimers could then bind the four sites presented in oriL. The same structure could be drawn for oris (asshown here) with the slight modification that one of the DNA-binding sites would be unoccupied or only loosely attached to DNA. The protein-protein interactions between the Nterminal part of the OBP molecules stabilize the structure. The ATPbinding site located in the N-terminal domain of OBP has been schematically depicted, and the C-terminal DNA-binding domain of OBP has been shaded.

1

'0

20

50

40

FIG.7. OBP but not AOBP binds cooperatively tooris. The scans were performed on the autoradiograph shown in Fig. 6. The dotted lines in all panels correspond to thescan of panel a. The solid lines refer to experiments where OBP or AOBPwereused. a, no protein added (4th lane);b, AOBP added (5th lane);c, OBP added (6th lane).

protein-protein contacts between OBP bound to boxes I and 111. Alternatively, binding to box I11 may depend on the previous binding of OBP toboxes I and 11, allowing for novel interactions between the C-terminal domains. According to the model presented in Fig. 8, the DNA helix is wrapped around the protein core, leaving the central ATrich region exposed to othermacromolecules. The conforma-

tion of this partof oris is changed as indicated by an altered DNaseI cleavage pattern and an enhanced sensitivity to micrococcal nuclease and potassium permanganate (Elias et al., 1990, Koff et al., 1991). The role of the spacer region is unknown. It may solelyexist to promote a correctand efficient formation of the nucleoprotein complex at oris. The initial unwinding would by analogy with other systems take place elsewhere (Kornberg and Baker, 1992; Borowiec et al., 1990). On the otherhand, a replacement of the AT-rich spacer with GC base pairs will completely abolish the ability to initiate DNA synthesis at oris. This would argue that conformational changes of the spacer region such as unwinding may be an essential property of the initiation process (Lockshon and Galloway, 1988).Experiments aimed at resolving this question are in progress. It is apparent from Fig. 8 that thebinding of nucleotides to the N-terminal part of OBP may affect the protein-protein interactions that stabilize the OBP-oriscomplex. We have so far failed to obtain reproducible results implying a role for ATP in the formation of OBP-oris complexes. We have, however, examined the influence of ATP and ADP on the mobility of OBP-oligonucleotide complexes in polyacrylamide gels. It was evident from these studies that ATP and ADP prevented the appearance of OBP-oligonucleotide aggregates on top of the gel and promoted the appearance of a more rapidly migrating species when the nucleotides were included

Protein-Protein Interactions an at in thegel at a concentrationof 5 mM.' A role for ATP binding and/or ATP hydrolysis in modulating the protein-protein interactions within the OBP-oriscomplex is therefore likely. As a finalcomment on the model presented above, we would like to emphasize that the actualnumber of OBP molecules within the nucleoprotein complex at the viral origins of replication may exceed the number of recognition sequences present in orisor oriL. We base thisstatement on the observation that OBP-oligonucleotide complexes can recruit free OBP dimers into large rapidly sedimenting aggregate^.^ In thisdiscussion, we have so far assumed that OBPis the only protein required for the activation of oris. In a way similar to E. coli and SV40, the initiatorprotein would provide the entry point for the remaining components of the replisome. Several observations do, however, argue that sequence elements outside the minimal oris, as well as factors bound t o these sequences can dramatically affect the ability of oriscontaining plasmids to replicate in cells superinfected with HSV-1 (Wong and Schaffer, 1991; Dabrowski and Schaffer, 1991). In our case, we have noted that the plasmid PORI Ib is more efficient in thetransient replication assay when compared with the plasmid pCG 5, indicating that minor alterationsinthe flanking sequences may have profound effects on replication.2 The view that additional factors may be required receives indirect support from a recent observation that the transcription activator E2 stimulates the binding of the E l replication protein to the minimal origin of replication of bovine papilloma virus and activates DNA synthesis (Yang et al., 1991). In the case of herpes simplex virus, both oriL and oris are located in transcriptionally active regions of the viral chromosome, and it has been shown that oris is surrounded by multiple Spl-binding sites (Jones andTjian, 1985). Inasmuch as Spl is involved in looping of DNA, one might imagine a situation where the simultaneous binding of OBP and Spl to the origin region might create complex structures in which distant sequence elements are brought closer to the origin (Su et al., 1991; Mastrangelo et al., 1991). 0. Hammarsten, unpublished observations. P. Elias, unpublished observations.

Origin of Replication

17429

The formation of these structures may also have topological consequences and thereby affect the helical stability of DNA. REFERENCES Arndt-Jovin, D. J., Jovin, T. M., Bahr, W., Frishauf, A,"., and Marquardt, M. (1975) Eur. J. Biochem. 64,411-418 Borowiec, J. A., Dean, F. B., Bullock, P. A., and Hurwitz, J. (1990) Cell 60,

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