Mechanism of stimulation of DNA replication by bacteriophage phi 29 ...

THEJOURNAL OF

BIOLOGICAL CHEMISTRY

Vol. 266, No. 4, Issue of February 5, pp. 2104-2111,1991 Printed in U.S A .

0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanism of Stimulation ofDNA Replication by Bacteriophage429 Single-stranded DNA-binding Protein p5* (Received for publication, July 6, 1990)

Crisanto GutierrezS, Gil Martins, Jose M. Sogoll, and Margarita Salasll From the Centro de B i o l o h Molecular ICSZC-UAMI. Canto Blanco, 28049 Madrid, Spain and the lllnstitutfur Zellbiologie, ETH-Honggerberg, CH-8093 Zurich, Switzerland ,1

Protein p5 is a Bacillus subtilis phage (629-encoded protein required for 429 DNA replication in vivo.Protein p5has single-stranded DNA binding (SSB) capacity and stimulates in vitroDNA replication severalfold when 429 DNA polymerase is used to replicate either the natural (629 DNA template or primed M13 singlestranded DNA (ssDNA). Furthermore, other SSB proteins, including Escherichia coli SSB, T4 gp32, adenovirus DNA-binding protein, and human replication factor A, can functionally substitute for protein p5. The stimulatory effect of (629 protein p5 is not due to an increase of the DNA replication rate. When both 629 DNA template and M13 competitor ssDNA are added simultaneously to the replication reaction, 429 DNA replication is strongly inhibited. This inhibition is fully overcome by adding protein p5, suggesting that protein p5-coated M13 ssDNA is no longer able to compete for replication factors, probably 429 DNA polymerase, which has a strong affinity for ssDNA. Electron microscopy demonstrates that protein p5 binds to M13 ssDNA forming saturated complexes with a smoothly contoured appearance and producing a 2fold reduction ofthe DNA length. Protein p5 also binds to ssDNA in the 429 replicative intermediates produced in vitro, which are similar in structure to those observed in vivo.Our results strongly suggest that (629 protein p5 is the 429 SSB protein active during 429 DNA replication.

minal protein (p3)covalently attached to its 5’-ends (Salas et al., 1978), has been studied extensively (reviewed in Salas, 1988). Genetic, biochemical, and electron microscopy studies have served to establish the general mechanism of 429 DNA replication i n uivo and to identify several gene products involved in thisprocess. The development of a very efficient in vitro system, which uses the 429 natural template (terminal protein-429 DNA complex), has allowed definition of the role of several phageencoded replication proteins (Salas, 1988). 429 DNA polymDNA replication by forminga erase(proteinp2)initiates covalent linkage between the phage-encoded terminal protein and the first dAMP residue (Blanco and Salas,1984; Watabe et al., 1984). $29 DNA polymerase then elongates the initial p3-dAMP complex in a very processive way until unit-length 429 DNA is formed (Blanco and Salas, 1985; Blanco et al., 1989). Concomitantly, the non-template strand is displaced as a ssDNA, giving rise to type I RI in which a dsDNA is in front of the DNA polymerase at thegrowing point. Complete displacement of a full-length ssDNA has been proposed for adenoviruses (Stillman, 1989; Kelly et al., 1988). However, in vioo studies (Escarmiset al., 1989) suggestthat initiation from both 429 DNA ends takesplace before displacement of a fulllength ssDNA occurs. When two growing chains runningfrom opposite ends collide, two type I1 RI are formed, which are characterized by the presenceof ssDNA in frontof the DNA polymerase. 429 DNA polymerase and terminal protein are the minimal protein requirements for 429 DNA replication i n vitro. HowInitiation mechanisms of DNA replication can use either a ever, other phage-encoded proteins are required i n vivo (renucleic acid or a protein as a primer. The two best known viewed in Salas, 1988). Recently, the gene coding for one of systems thatuse a protein-priming mechanism are theB u d - those proteins, p5, has been cloned, and protein p5 hasbeen lus subtilis bacteriophage 429 and theadenoviruses. Replica- overproduced (Martin and Salas, 1988), purified, and shown tion of 429 DNA, whose genome is a 19285-base pair-long to have affinity for ssDNA and to stimulate 429 DNA replilinear dsDNA’ molecule (Vlcek and Paces, 1986) with a ter- cation i n vitro (Martin et al., 1989).Consequently, it was postulated that p5 might participate as a SSB protein during * This investigation has been aided in part by Research Grant 5 429 DNA replication. R01 GM27242-11 from the National Institutes of Health, by Grant Proteins that bind ssDNA to with high affinity but without PB87-0323 from Direccibn General deInvestigacionCientificay Tbcnica, by an institutional grant from Fundacih Ramon Areces, sequence specificity have been purified from several sources to (Chase andWilliams, 1986).In addition to binding ssDNA, and by Grant 31-25178.88 from the Swiss National Foundation. The costs of publication of thisarticle were defrayed in part by the to be considered as SSB replication proteins, they should payment of page charges. Thisarticlemusttherefore be hereby participate in DNA replication. For example, they may stimmarked “aduertisement” in accordance with 18 U.S.C. Section 1734 ulate their cognateDNA polymerases in stoichiometric quansolely to indicate this fact. $ Supported by a European Molecular Biology short term fellow- tities with respect to the template, or have an essential role ship andby a fellowship from the Ministerio de Educacibn y Ciencia at some stage during genome replication, as is the case for phage fd protein 5 , which controls the switch from RF to of Spain. Present address: Antibioticos S.A., Antonio L6pez 109-111,28026 replication of viral ssDNA (Kornberg, 1980). Madrid, Spain. Inthis work we haveanalyzed furtherthefunction of 1) To whom correspondence should be addressed. protein p5 during 429DNA replication by carrying out both ’ The abbreviations used are: dsDNA, double-stranded DNA; SSB, biochemical and electronmicroscopy studies (i) to understand single-stranded DNAbinding; ssDNA,single-strandedDNA;RI, replicative intermediate; Ad, adenovirus, DBP, DNA binding protein; the mechanism of stimulation of DNA replication by protein p5 and (ii) to characterize the interactionof protein p5 with RF-A, replication factor A.

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Stimulation of DNA Replication by $29 SSB Protein ssDNA in both the RI produced during in vitro 429 DNA replication and i n the protein p5-Ml3 ssDNA complexes. that protein p5 is the SSB Altogether, our results suggest protein active during 429DNA replication and that its major mechanism of stimulation of 429 DNA replication in vitro is t o increase the number of reinitiations on new templatesas a result of facilitating recycling of 429 DNA polymerase molecules. EXPERIMENTALPROCEDURES

Materials The $29 DNA polymerase p2, the terminal protein p3, and protein p5 were purified by J. M. Lazaro (Centro deBiologia Molecular, Spain), as described (Blanc0 and Salas, 1984; Prieto etal., 1984; Martin et al., 1989). 429 DNA-protein p3 complex was prepared as described (Peiialva and Salas, 1982). Escherichia coli SSB protein, T4 gp32, Ad DBP, and human RF-A were obtained from A. Kornberg (Stanford University), J.-J. Toulme (Fondation Edmond de Rostchild, France), P. van der Vliet (State University of Utrecht, the Netherlands), and B. Stillman (Cold Spring Harbor Laboratory), respectively. The following reagents were commercially available: proteinase K and unlabeled deoxynucleotidesfrom Boehringer Mannheim, [ ( u - ~ * P ] ~ A(-400 T P Ci/mmol) from Amersham International, and M13mp18 ssDNA from U. S. Biochemical Corp.

Methods ReplicationAssaywith 429 DNA-Terminal Protein Complex as Template-For $29 DNA-terminal protein replication, the incubation mixture contained, unless otherwise stated, in 20 pl, 50 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 1 mM dithiothreitol, 10 mM ammonium sulfate, 25 pg of bovine serum albumin, 40 p M each dCTP, dTTP, dGTP, and [(u-~’P]~ATP (1 pCi), 220 ng of$29 terminal protein-DNA complex, 25 ng of 429 DNA polymerase p2, and 15 ng of $29 terminal protein p3. Purified protein p5 (6 pg) wasadded when indicated. After different times at 30 “C, reactions were stopped by adding EDTA and sodium dodecyl sulfate to 10 mM and 0.1%, respectively. The samples were filtered through Sephadex G-50 spun columns in the presence of 0.1% sodium dodecyl sulfate to remove unincorporated dNTPs.The Cerenkov radiation of the excluded volume was then determined. To determine the size of newly synthesized DNA,replication products were fractionated by gel electrophoresis in 0.6% agarose alkaline gels, and the autoradiograms were scanned in a computing densitometer, model 300A (Molecular Dynamics) with the aid of ImageQuant software. Replication Assay with Primed M I 3s s D N A as TemplateM13mp18 ssDNA (300 ng) was hybridized with a 17-mer oligonucleotide primer (10 ng) in an incubation mixture containing, in 4 p l , 16.7 mM Tris acetate, pH 7.5, 75 mM NaC1, by heating at 56 “cfor 15 min and then coolingdown to 30 “C, slowly. The replication reaction mixture contained, in 10 pl, 50 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 1 mM dithiothreitol, 10 mM ammonium sulfate, 10pgof bovine serum albumin, 40 p~ each dCTP, dTTP, dGTP, and [(u-’~P]~ATP (1 pCi), 4 pl of the solution containing the primed M13 ssDNA template, as described above, and 12.5 ngof $29 DNA polymerase. After incubation at 30 “C for the indicated times, reactions were stopped and the samples processed as described in the previous section. Psoralen Cross-linking and Spreading of D N A Molecules for Electron Microscopy-To analyze the structure and type of replicative intermediates produced during $29 DNA replication in vitro, replication reactions were stopped by adding 0.05 volume of 4,5’,8-trimethylpsoralen (200 pg/ml in 100% ethanol) on ice. Samples were then irradiated with ultraviolet light on ice as described by Sogo et al. (1984). Psoralen was added three more times during atotal irradiation time of 4 h. After psoralen cross-linking, the samples were digested with proteinase K (500 pg/ml) for 2 h at 50 “C andextracted with phenol, and the DNA was precipitated with ethanol. Denaturation and spreading of the psoralen-cross-linked DNA for electron microscopy were performed as described by Sogo et al. (1984). Electron Microscopy of Protein-DNA Complexes-To analyze the interaction of protein p5 with DNA in the replicative intermediates produced during in vitro 629 DNA replication, DNA-protein complexes were separated away fromfree protein by gel filtration through a Sepharose CL-4B column equilibrated with 7 mM magnesium

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acetate. Fractions were collected on tubes containing glutaraldehyde to give a final concentration of 0.1%. After 1 h at room temperature, samples were prepared for electron microscopy according to theBAC (benzyldimethylalkylammonium chloride) spreading technique (Sogo et al., 1987). In some experiments E. coli SSB protein was used instead of protein p5, and the samples were processed in the same way. To analyze the interaction between protein p5 and M13 ssDNA they were incubated, at a mass ratio of 401, in the same buffer used for in vitro 429 DNA replication. After 20 min at 30 “C,DNA-protein complexeswere purified as described in the previous paragraph. Single-stranded M13 DNA was added just before spreading and was used as an internalcontrol for contour length measurements. Samples were spread as described (Sogo et al., 1987). Micrographs were taken in a Philips 420 electron microscope at 100 kV (magnification, X 18,500). Contour length was determined in a Hewlett-Packard digitizer. RESULTS

Stimulation of DNA Replication by 429 Proteinp5 and Other SSB Proteins-Previous experiments indicated that protein p5 has a strong affinity forssDNA, suggesting that p5 could act as a SSB protein during 429DNA replication (Martin et al.,1989). The extent of DNA replication was determined under standard in vitro replication conditions either with or without $29 p5 or E. coli SSB, a well characterized SSB protein (Fig. 1A). As previously reported (Martin et al., 1989), p5 greatly stimulates 429DNA replication, especially at long incubationtimes,whenreactionslackingproteinp5have reached a plateau. At similar protein concentrations, E. coli SSB was also able to stimulate 629 DNA replication carried out by 429DNA polymerase. In both cases the kinetics were very similar witha small effect upto 5 min reaction time and a significant stimulation (up to 4.5-fold) at longer reaction times (20-60 min). This stimulatory effect wasalso observed when other SSB proteins were used instead of p5, including T 4 -32, Ad DBP, and human RF-A (datanotshown). Therefore, the fact t h a t E. coli SSB (as well as other SSB 300

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FIG. 1. Stimulation of DNA replication by 429 protein p5 and E. coli SSB protein. A, $29 DNA template was incubated with purified 429 DNApolymerase and $29 terminal protein in the absence (open circles) or in the presence of protein p5 (closed circles) or E. coli SSB protein (closed squares) at 30 “C for the indicated times, and the extent of DNA replication was determined as described under “Methods.” B , as in A , but primed M13 ssDNA was used as atemplate primer instead of 429 DNA template.

Stimulation of DNA Replication by 429 SSB Protein

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FIG.2. Effect of 429 protein p5 on the rate of DNA replication. A, @29 DNAtemplate was incubated with purified 429 DNA

of protein p5 (Fig. 2 A ) . A similar experimentwas carried out using primed M13 ssDNA as a template. In this case, there was no apparentlag in the initiationreaction, and protein p5 produced a %fold stimulation of the dNMP incorporation. Again, this stimulation was not the result of an increase in the DNA elongation rate (Fig. 2B). Therefore, those results showed that thelarge stimulatory effect of 429 protein p5 on DNA replication cannot be explained by an increase in the DNA replication rate either on the 429 natural template or on primed M13 ssDNA. Inhibition of 429 DNA Replication by Competitor MI3 ssDNA Is Overcome by 429 Protein p5"During 429 DNA replication increasingamounts of ssDNA are produced. Since 429 DNA polymerase has a strong affinity for ssDNA (Blanco et al., 1989), the possibility exists thatprotein p5 stimulates 429 DNA replication by blocking nonproductive binding of 429 DNA polymerase to ssDNA. Therefore, we carried out a replication assay in which increasing amounts of competitor M13 ssDNA were added to the in vitro replication reaction (Fig. 3). When both template and competitor ssDNA were added simultaneously to a reaction mixture lacking protein p5, 429 DNA replication was strongly inhibited. However, if competitor M13 ssDNA was preincubated with protein p5 and then added to the replication reaction, 429 DNA replication was essentially unaffected. Control experiments using dsDNA as competitor showed that inhibition of 429 DNA replication was specific for ssDNA. This result indicated that naked but not proteinp5-coated M13 ssDNA was competing for replication factors (probably 429 DNA polymerase) necessary for both initiation and elongation of 429 DNA templates. This idea was reinforced by subsequent competition experiments in which both 429 DNA polymerase and 429 DNA template were preincubated before either naked or protein p5-coated M13 ssDNA were added to thereplication reaction (Fig. 3). Under these conditions, M13 ssDNA was a poor competitor, consistent with the fact that 429 DNA polymerase is a highly processive enzyme. These results suggest that prevention of nonproductive binding of 429 DNA polymerase to the single-stranded portions of 429 RI might

polymerase and @29terminal protein in the absence(open circles) or in thepresence (closed circles) of protein p5a t 25 "C for the indicated times. Replication products were fractionated by alkaline agarosegel electrophoresis (inset), and the rate of DNA replication was determined as described under "Methods." B, as in A, but primed M13 ssDNA was used as a template primer insteadof 429 DNA template. nt, nucleotides.

proteins) can functionally substitute for p5 during 429 DNA replication suggests that the mechanism of stimulation of DNA replication could be common to all these SSB proteins, including 429 protein p5. Since $29 DNA polymerase is able to carry out DNA synthesis using singly primed M13 ssDNA (Blanco et al., 1989), we also analyzed the effect of protein p5 on this429 DNA polymerase-dependent DNA replication. As shown in Fig. lB, both 429 p5 and E. coli SSB stimulated DNA synthesis carriedout by 429 DNA polymerase. Although this stimulatory effect was greater a t long reaction times (2060 min), it was also apparent at short reaction times (2-5 min). To determine whether 429 protein p5 had any effect on the DNA elongation rate, replication products formed a t short reaction times were fractionated by alkaline agarose gel electrophoresis, and the average size of newly synthesized DNA was estimated by densitometric scanning of the autoradiogram. Using the natural 429 DNA template, the DNA replication rate was 4100 nucleotides/min in the absence of 429 protein p5, about 75% of the value obtained in the presence

5

1 0 50 100 150 20( Competitor M13 ssDNA (ng)

FIG.3. 429 DNA replication in the presence of competitor M13 ssDNA. The effect of competitor M13 ssDNA added beforethe formation of the preinitiation complex was measured by incubating @29DNA polymerase and $29 terminal protein simultaneouslywith the @29 DNA template and competitor naked M13ssDNA(open circles) or proteinp5-coated M13 ssDNA (closed circles). Competition after initiation complex formation was tested by preincubating 429 DNA polymerase and 629 terminal protein with the $29 DNA temM13 ssDNA plate for 5 min on ice and then adding competitor naked (open squares) or protein p5-coated M13 ssDNA (closed squares). As a control, dsDNA (from plasmid pUC19) was used (triangles). In all cases, reactions continued for 60 min a t 30 "C.

Stimulation of DNA Replication by 429 SSB Protein

FIG.4. 629 replicativeintermediates produced in vitro. Replication reactions with $29 DNA polymerase, $29 terminal protein, and $29 DNA template were incubated a t 30 “C during 20 min in the absence ( A ) or in the presence ( B ) of protein p5 and then treated with psoralen under UV irradiation. Reaction products were then digested with proteinase K and extracted with phenol, and the DNA was purified by ethanol precipitation. Samples were prepared for electron microscopy as described under “Methods.” Bar represents 0.5 pm. Arrows point to the displaced ssDNA in a two-tailed type I RI ( A ) and in a three-tailed type I ( B ) .

be the primary mechanism by which protein p5 stimulates 429 DNA replication in vitro. Protein p5 Protects the Displaced ssDNA from Psoralen Cross-linking-Binding of protein p5 to ssDNA protects the latter against nuclease P1 digestion, suggesting a possible role for protein p5 in the elongation step by interaction with the displaced ssDNA being produced (Martin etal., 1989). It has been demonstrated that psoralen cross-links the portions of the DNAmolecule that are free of proteins, not only in dsDNA, i.e. linker DNA inchromatin (Sogo andThoma, 1989), but also in the folded-back regions in single-stranded nucleic acid molecules (Wollenzien, 1988).This procedure has been successfully applied to theanalysis of both transcription and replication (Sogo et al., 1984, 1986;Lucchini et al., 1987). Therefore, if protein p5 interacts with ssDNA during 429 DNA replication, it could prevent the displaced ssDNA from psoralen cross-linking. Consequently, in vitro replication reactions carried out both inthe absence and presence of protein p5 were treated with psoralen as described under “Methods.” The reaction products were then extracted with phenol and the DNA products purified and spread as described under “Methods.” Electron microscopic observations revealed that several types of RI molecules were present during 429 DNA replication in vitro: full-length dsDNA molecules with one or more ssDNA tails (type I RI) andfull-length DNA molecules formed by a dsDNA portion of variable length from one DNA end plus a ssDNA portion spanning to the other DNA end (type I1 RI). In some cases, these type I1 RI also contained one or more ssDNA tails emerging from the dsDNA portion (type 1/11 RI). Thus, thepredominant types of intermediate molecules corresponded with those found in vivo (Inciarte et al., 1980; Sogo et al., 1982), indicating that the in vitro 429 DNA replication conditions used mimic the in vivo situation. A detailed kinetic study of RI produced during 429 DNA replication in vitro will be presented elsewhere.‘ The absence or presence of protein p5 during in vitro 429 DNA replication had a clear effect on the structure of such RI molecules after psoralen cross-linking. The ssDNA por-

’C. Gutikrrez, J. M. Sogo, and M. Salas,

manuscript in preparation.

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tions of RI produced in the absence of protein p5 collapsed, presumably because the cross-links introduced by psoralen in the folded-back portions of ssDNA stabilized their secondary structure, preventing their complete unfolding (Fig. 4A). In contrast, ssDNA found in RI molecules produced in the presence of p5 had a well unfolded structure (Fig. 4B). Moreover, it is known that addition of protein p5 30 min after replication has started,when DNA replication essentially has leveled off, stimulates DNA replication to an extent similar to thatobtained when protein p5is present from the beginning (Martin et al., 1989).Therefore, we tried todetermine whether protein p5 prevented ssDNA in RIfrom psoralen cross-linking when it was added to a reaction mixture that had been kept for 30 min a t 30 “C in the absence of p5. Under those conditions, ssDNA portions of RI molecules found 5 min after addition of protein p5 had an extended appearance, indistinguishable from those found in reactions carried out in the presence of protein p5 from the beginning (data not shown). Altogether, these results led us to conclude that protein p5 not only protects ssDNA against nuclease digestion but also prevents ssDNA from psoralen cross-linking, suggesting strongly that p5 interacts specifically with the ssDNA portions of RI molecules during 429 DNA replication. Structure of Protein p5-RI DNA Complexes Produced during in Vitro 429 DNA Replication-To determine the structure of the complexes formed between protein p5 and ssDNA portions of RI molecules, protein p5-DNA complexes were prepared for electron microscopy by the glutaraldehyde crosslinking procedure (Sogo et al., 1987). Since E. coli SSB protein was able to substitute functionally for p5 during 429 DNA replication (see Fig. l),E. coli SSB was also included, as a control, in some experiments. Replication reactions were carried out both in the absence and presence of either E. coli SSB or p5. After 20 min at 30 “C, purified DNA-protein complexes were treated with glutaraldehyde and analyzed by electron microscopy. In both cases the effect of the fixation time (before, during, or after theSepharose column) was also determined. $29 protein p5-DNA complexes were best visualized when glutaraldehyde fixation took place after purification of the complexes through the Sepharose column. Fixation before this stepproduced large aggregates, which could not be properly analyzed (datanot shown). The time of fixation had no significant effect on either theyield or structure of E. coli SSB-ssDNA complexes. Under the replication conditions used (input 429 DNA to protein p5 mass ratio was 1:30), protein p5was able to interactwith the ssDNA portions of the different types of 429 RI (Fig. 5 A-C). However, the structure of the p5-DNA complexes was completely different from that found with E coli SSB (Fig. 5D), which forms a more compact structure. Quantitations of the different types of proteinp5-RI complexes showed that 65.4 and 34.6% corresponded to types I and I1 RI, respectively. In thecase of p5 complexes, small ssDNA loops appeared scattered over the ssDNA (Fig. 5C, arrows) suggesting that interaction of p5 with ssDNA did not occur over the entirelength of the ssDNA. Essentially the same results were obtained when the protein p5 to DNA ratio was increased 2-fold and when formaldehyde (0.2% final concentration)was used instead of glutaraldehyde as a cross-linking agent (data not shown). Alternatively, the small ssDNA loops observed could be the result of a loss of part of protein p5 during purification and fixation of the complexes. This explanation is in agreement with the fact that ssDNA in RI molecules produced in the presence of protein p5 appeared fully extended after psoralen cross-linking (see Fig. 4B). Nevertheless, we conclude that protein p5 binds to the ssDNA portions of RI molecules during in vitro

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Stimulation of DNA Replication by 429 SSB Protein

FIG. 5. Interaction of 429 protein p5 and E. coli SSB with ssDNA in 429 replicative intermediates produced in vitro. Replication reactions using 629 DNAas template were carried outas described in Fig. 4 in the presence of $29 protein p5 ( A - C ) or E . coli SSR protein (11) at 30 “C for 20 min. The DNA-protein complexes were purifiedby pel filtration through Sepharose columns and treated with 0.1% glutaraldehydeat 30 “C for 1 h. Samples were prepared for of electronmicroscopy as describedunder“Methods.”Complexes protein p.5 with ssDNA (thick curtled arroux) in a type I (A ), a type 1/11 ( R ) , and a type I1 (C) RI molecule, and of E. coli SSR protein with ssDNA in a type I1 RI ( D ) are shown. Thin arroux in C point t o protein-free ssDNA regions. Bar represents0.5 pm.

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FIG. 6. Complexes of 429 protein p5 andE. coli SSR protein with M13 ssDNA. A, purified p5 was incubated for 20 min at 30 “C 429 DNA replication. This, together with previous observawithM13ssDNA at a protein/DNAratio of 40:l.DNA-protein tions, demonstrates that protein p5 acts as a SSB protein complexes were purified and visualized under the electron microscope during 429 DNA replication. as described under “Methods.” Two protein p5-Ml3 ssDNA comProtein p5 Reduces the Length of ssDNA in the p5-ssDNA plexes (arrows) and one M13 ssDNA molecule used as an internal Complex-It is known that interaction of SSB proteins with control are shown. R, same as A hut E. coli SSR protein was used ssDNA leads to a change in the ssDNA length. In some cases, instead of $29 protein p5. One SSR-M13 ssDNA complex (arrow) are shown. Bar represents 0.5 pm andoneM13ssDNAmolecule suchas in the case of E. coli SSB,thereis aseveralfold (1215 nucleotides). C, contour length measurements of protein-free reduction in length upon formation of the complex (Griffith M13 ssDNA (white histogram), protein p5-Ml3 ssDNA complexes et al., 1984). Thus, it was interesting to determine whether (black histogrum), and E . coli SSR protein-M13 ssDNA complexes of each size interactions of p5 with ssDNAalso produced a change in the (shaded histogrum). Arrows point to the mean values ssDNA length. T o obtain a ssDNA of a defined structure and distribution (275.3, 159.0, and 70.6 units for protein-free DNA, p5M13, and SSR-Ml3 complexes, respectively). A total of 64, 71, and length, M13 ssDNA was incubated under in uitro 429 DNA 34 molecules was measured in each case. Fifty units correspond to replication conditions, with either p5 orE. coli SSB (used as 1350 nucleotides.

a control). DNA-protein complexes were then purified, fixed with glutaraldehyde, and spread for electron microscopy as described under“Methods.”Protein-free M13 ssDNA was also included just before thespreadingto be used as an internal control for both the spreading procedure and the contour length measurements. Under the experimental conditions used, both p5 and E. coli SSB efficiently associated withM13ssDNA,coveringcompletely the DNA molecule (Fig. 6, A and B , respectively). As expected, in the case of E. coli SSB protein, saturatedcomplexes were found with almost no ssDNA regions free of protein in the complex. This was also the case for most of the protein p5-Ml3 ssDNA complexes. However, in the latter case,a few molecules were found to contain some protein-free ssDNA loops of variable length, similar to those described for the RI. When formaldehyde (0.2% final concentration) was used instead of glutaraldehyde to stabilize protein-DNA complexes, essentially the same results were obtained (data not shown). The possible significance of such loops has not been studied further. The structure of protein p5-Ml3 ssDNA complexes had a smoothly contoured appearance as compared with the more compact structure of E. coli SSB protein-M13ssDNA complexes (Fig. 6, A and R). Simple inspection of thesampleunderthe electron microscope indicated that interactionof p5 with M13

ssDNA produced a significant reduction in the DNA length (Fig. 6 A ) . This reduction was, however, clearly smaller than that achieved with E. coli SSB (Fig. 6 R ) . T o quantitate the extent of length reduction upon complex formation, the contour length of both protein-free andE. coli SSB- and 429 p5M13 DNA complexes was determined. As shown in the histograms of Fig. 6C, under optimal conditions for 429 DNA replication, the length of the E. coli SSB-Ml3 ssDNA complexes was reduced about 4-fold compared with protein-free M I 3 ssDNA, in agreement with previous findings (Griffith et al., 1984). Under the same conditions, association of protein p5 with M13 ssDNA produced a saturated complex with a length about 2-fold smaller than the corresponding ssM13 protein-free DNA. DISCUSSION

Mechanism of Stimulation of D N A Replication in Vitro by protein p5 shares several of the properties exhibited by SSB proteins. It is an essential component during 429 DNA replication in uiuo (Mellado et al., 1980) and greatly stimulates in uitro $29 DNAreplication in assays using either 429 terminal protein-DNA complex or primed M13 ssDNA. Furthermore,stoichiometricamounts of the 429 Protein p5-429

Stimulation of DNA Replication by 429 SSB Protein

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3--R

FIG. 7. The ssDNA binding motif. Partial amino acid sequences from several prokaryotic and eukaryotic proteins with known affinity for ssDNA have been aligned as previously described (Prasad andChiu, 1987; Wang and Hall, 1990), including $29 protein p5. Amino acids are shown by single-letter abbreviations, and thesubscripts indicate the position in the sequence of the first and last residues. The number of residues present inthe unrelated spacer regions is also shown. The positions corresponding to aromatic residues have been shaded. Boxes point to amino acids implicated in binding to ssDNA (see "Discussion" for details). In addition to the sequence F-27K-3°F in $29 protein p5 shown in this figure, the sequence F-25-R-5-F occurs in the same region. Sequences shown are from prokaryotic (E. coli SSB (Sancar et aL, 1981), phage fd gp5 (Cuypers et a[., 1974; Nakashima et al., 1974), phage Ike gp5 (Peeters et al., 1983), phage T4 gp32 (Williams et al., 1981), and phage $29 SSB protein p5 (Martin and Salas, 1988)) and eukaryotic (Ad DBP (Kruijer et al., 1981), herpes simplex virus-1 ( H S V - I )ICP-8 (McGeoch et al., 1988),varicella zoster virus ( V Z V )DBP (Davison and Scott, 1986), and EpsteinBarr virus ( E B V ) DBP (Baer et al., 1984)) proteins.

protein with respect to thetemplate are required for efficient stimulation (Martin et al., 1989). Protein p5 binds preferentially to ssDNA protecting it from nuclease ( i e . nuclease PI) digestion (Martin et al., 1989), and high salt is required to dissociate the complex (Martin et al., 1989). Once a protein has met those criteria (Chase and Williams, 1986), it should be considered as a bona fide SSB protein and, consequently, as in the case ofwell known SSB proteins, p5 should be considered as the429 SSB protein. Several mechanisms may explain the stimulatory effect of DNA replication by SSB proteins. Some of them seem to involve a specific interaction of the SSB protein with its cognate DNA polymerase, as in thecase of bacteriophage N4 SSB (Lindberg et al., 1989), yeast stimulatory factor (Brown et al.,1990),Ad DBP (Lindebaum et al.,1986, herpes simplex virus 65K DBP (Gallo et al., 1989), and human SSB (Kenny et al., 1989). In these cases, SSB proteins may modulate the processivity of the DNA polymerase or its associated 3'3'exonuclease activity. On the other hand, a nonspecific stimulation of DNA polymerase activity by SSB proteins has also been observed in some DNA replication systems, as in the case of human DNA polymerase 6 activity, using (dA)rooo . (dT)12-18 as a primer template and several prokaryotic and eukaryotic SSB proteins (Kenny et al., 1989). In these cases, SSB proteins may modulate DNA polymerase activity indirectly either by increasing the affinity of DNA polymerase for primer ends, by overcoming the inhibitory effect of ssDNA, or by facilitating recycling of DNA polymerase molecules after termination. Moreover, E. coli SSBproteinstimulates T7 DNA polymerase by both specific and nonspecific mechanisms (Myers and Romano, 1988). Since 429DNA replication was stimulated not only by protein p5 but also by E. coli SSB as well as other SSB proteins such as T4 gp32, Ad DBP, and human RF-A, the stimulatory effect of DNA replication by protein p5 appears to be nonspecific. We found that protein p5 had only a small stimulatory effect on the DNA replication rate using both the natural 429 DNA template and primed M13 ssDNA, which represent model templates for type I and type I1 RI, respec-

tively. This small stimulatory effect cannot account for the severalfold increase in the total dNMP incorporation, especially at long incubation times. Furthermore, 429 DNA replication is strongly inhibited by M13 ssDNA in challenger competitor-template experiments. However, once 429 DNA polymerase interacts with the natural 429 DNA template, M13 ssDNA is no longer able to strongly inhibit 429 DNA replication. It is known that in the in vitro 429 DNA replication system, in the absence of protein p5, input DNA mass is roughly doubled when the plateau is reached (Blanco et al., 1989), suggesting that only two initiation events haveoccurred. It is also known that stimulation of 429 DNA replication by protein p5 does not occur at thelevel of the initiation complex formation, although protein p5 increases the number of reinitiations at long incubation times (Martinet al.,1989). Therefore, we propose the following major mechanism of stimulation of DNA replication in vitro by protein p5. 429 DNA polymerase efficiently initiates DNA replication on the natural terminal protein-DNA template in a protein p5-independent way. It is then able to further elongate this initial complex very processively.As this occurs, increasing amounts of ssDNA, produced as a consequence of displacement of the non-template strand, sequester DNA polymerase molecules, thus inhibiting reinitiation events on new templates. Covering of ssDNA in RI by protein p5 would overcome inhibition by ssDNA and would facilitate efficient recycling of DNA polymerase molecules, which, upon termination of DNA replication, could reinitiate onnew templates. In addition, the possibility, which is now under study, that protein p5 could have also an effect at other levels during 429 DNA replication, such as facilitating the termination stage, cannot be ruled out at this time. The Complex of 429 Protein p5 with ssDiVA-The properties discussed above are the result of an interaction between protein p5 andssDNA leading to theformation of a saturated ssDNA-protein p5 complex. Upon complex formation, protein p5 is able to protect the ssDNA from psoralen cross-linking and nuclease digestion. Electron microscopic analysis of DNA-protein complexes showed that both E. coli SSB and

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Stimulation of DNA Replication by $29 SSB Protein

429 p5 efficiently associated with the ssDNA portions of RI produced in uitro, as was already demonstrated for Ad DBP interacting with ssDNA in Ad RI produced in vivo (Kedinger et al., 1978).However, the structure of the DNA-protein complexes was clearly different. While E. coli SSB formed a tight complex giving a rodlike appearance to the ssDNA in RI, thecomplex of 429 protein p5 with ssDNA in RI exhibited a rather smooth structure without producing a large thickening of the ssDNA portion of the molecule. We have shown that several SSB proteins are interchangeable in the in uitro 429 DNA replication assay, in spite of the very different structures that they produce upon association with ssDNA in RI. This reinforces the idea that the major mechanism of stimulation of$29 DNA replication by p5 (or otherSSB proteins) is related to its ability to relieve the inhibition of the DNA polymerase activity by the increasing amount of ssDNA being produced during the reaction, as discussed above. Electron microscopic studies have shown that Ad DBP keeps the ssDNA in an extended form (van der Vliet et al., 1978) in such a way that 4x174 ssDNA associated with Ad DBP produces a complex whose length is about 15% smaller than the corresponding protein-free 4x174 dsDNA. E. coli SSB produces about a 3-4-fold reduction in length upon complex formation depending on the protein/DNA ratio (Griffith et al., 1984). In the case of protein p5, association with M13 ssDNA also produces a significant reduction (about 2-fold) in the M13 ssDNA length, suggesting that ssDNA wraps around protein p5 monomers, as hasbeen demonstrated for other SSB proteins (Chase and Williams, 1986; Lohman et al., 1988). In contrast, association of T4 gp32 with 4x174 ssDNA produces about a 40% increase in the DNA length (Delius et al., 1972). These structural differences are likely the result of different modes of binding (Lohman et al., 1988). Nevertheless, the existence of a consensus motif in SSB proteinsthat could be responsible for ssDNA binding has been postulated previously (Prasad and Chiu, 1987; Wang and Hall, 1990). This was based on the best alignment of aromaticand positively charged residues in a number of SSB proteins, which otherwise lack a strong amino acid homology (Fig. 7). As indicated in Fig. 7, $29 protein p5 exhibits such an array of functional residues potentially critical for ssDNA binding. For some of these proteins, spectroscopic studies, chemical modifications, and site-directed mutagenesis indicated that certain residues (frequently aromatic and positively charged) in the ssDNA binding motif are critical for ssDNA binding. It should be kept in mind, however, that aromatic residues need not be the only type involved in ssDNA binding. This seems to be the case in fd gp5 where, in addition to Tyr26 and Phe73,a leucine residue (Leu28)also seems to interact with oligodeoxynucleotide bases (King and Coleman, 1987, 1988). Furthermore, Tyre4, Tyrg9,Tyrlo6,and Tyr1I5in T4 gp32 (Prigodich et al., 1986; Shamoo et at., 1989), Trp5*and Phe6' in E. coli SSB (Casas-Finet et al., 1987; Khamis et al., 1987a, 1987b), and Phe469and Lys470in Ad DBP (Quinn and Kitchingman, 1986; Neale and Kitchingman, 1990) have been implicated in binding to ssDNA. Predictions of secondary structure for some of these SSB proteins showed that @-sheets and @-turns are their exclusive (fd gp5) or predominant (T4 gp32) structural feature. The predicted secondary structure of 429 protein p5 (Martin and Salas, 1988) indicates that it also has most of the molecule formed by @-sheets.Moreover, ts5(219) (529 mutant shows a single nucleotide change where a GGG triplet coding for a glycine residue (Gly7*)has been changed to a GAG triplet

coding for a glutamic acid residue (Martin and Salas, 1988). Predictions indicate that this amino acid change would convert a @-turn in the wild-type protein into a random coil in the ts5 protein, producing a major change in the structure of the mutant protein. Therefore, with the data available for fd gp5 and IKe gp5 (De Jong et al., 1989a, 1989b), it seems reasonable to speculate that similar protein-ssDNA interactions also exist in the 429 protein p5-ssDNA complex. However, future studies will be necessary to prove whether the conserved residues in 429 protein p5 are involved directly in ssDNA binding, as discussed above for some SSB proteins. Acknowledgments-We are grateful to Dr. T. Koller for his interest and support and to Drs. A. Kornberg, J.-J. Toulmi., P. van der Vliet, and B. Stillman, who kindly provided us with samples of purified E. coli SSB, T4 gp32, Ad DBP, andhuman RF-A, respectively. REFERENCES Baer, R., Bankier, T., Biggin, M. D., Deininger, P. L., Farrel, P. J., Gibson, T. J., Hatfull, G., Hudson, G. S., Satchwell, S. C., Seguin, C., Tuffnell, P. S., and Barrel], B. G. (1984) Nature 3 1 0 , 207-211 Blanco, L., and Salas, M. (1984) Proc. Natl. Acad. Sci. U. S. A . 8 1 , 5325-5329 Blanco, L., and Salas, M. (1985) Proc. Natl. Acad. Sci. U. S. A . 82, 6404-6408 Blanco, L., Bernad, A., Lazaro, J. M., Martin, G., Garmendia, C., and Salas, M. (1989) J. Biol. Chem. 264,8935-8940 Brown, W. C., Smiley, J. K., and Campbell, J. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,677-681 Casas-Finet, J. R., Khamis, M. I,, Maki, A. H., and Chase, J. W. (1987) FEBS Lett. 220,347-352 Chase, J. W., and Williams, K. R. (1986) Annu. Reu. Biochem. 55, 102-136 Cuypers, T., van der Ouderaa, F. J., and de Jong, W. W. (1974) Biochem. Biophys. Res. Commun. 59,557-563 Davison, A. J., and Scott, J. E. (1986) J. Gen. Virol. 67, 1759-1816 de Jong, E. A. M., van Duynhoven, J. P. M., Harmsen, B. J. M., Konings, R. N. H., and Hilbers, C. W. (1989a) J. Mol. Biol. 2 0 6 , 119-132 de Jong, E. A.M., van Duynhoven, J . P. M., Harmsen, E. J. M., Tesser. G. I.. Konines. R. N. H., and Hilbers, C. W. (1989b) J. Mol. Biol. 206,133-152Delius, H., Mantell, N. J., and Alberts, B. (1972) J. Mol. Biol. 6 7 , 341-350 Escarmis, C., Guirao, D., and Salas, M. (1989) Virology 169, 152160 Gallo, M.L., Dorsky, D. I., Crumpacker, C. S., and Parris, D. S. (1989) J. Virol. 63,5023-5029 Griffith, J., Harris, L. D., and Register, J. C., 111 (1984) Cold Spring Harbor Symp. Quant. Biol. 49,553-559 Inciarte, M. R., Salas, M., and Sogo, J. M. (1980) J . Virol. 34, 187199 Kedinger, C., Brison, O., Perrin, B. F., and Wilhelm, J. (1978) J. Virol. 2 6 , 364-379 Kelly, T. J., Wold, M. S., and Li, J. (1988) Adu. Virus Res. 34, 1-42 Kenny, M. K., Lee, S.-H., and Hurwitz, J. (1989) Proc. Natl. Acad. Sei. U. S. A . 86,9757-9761 Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Murphy, J . B., and Chase, J. W. (1987a) J. Biol. Chem. 262, 10938-10945 Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Murphy, J. B., and Chase, J. W. (1987b) FEBS Lett. 211,155-159 King, G. C., and Coleman, J. E. (1987) Biochemistry 26,2929-2937 King, G. C., and Coleman, J. E. (1988) Biochemistv 27,6947-6953 Kornberg, A. (1980) DNA Replication, pp. 489-494, W. H. Freeman & Co., New York Kruijer, W., van Schaik, F. M., and Sussenbach, J. S. (1981) Nucleic Acids Res. 9,4439-4457 Lindberg, G., Kowalczykowski, S. C., Rist, J. K., Sugino, A., and Rothman-Denes, L. B. (1989) J. Biol. Chem. 2 6 4 , 12700-12708 Lindebaum. J . 0.. Field., J... and Hurwitz, J. (1986) J. Biol. Chem. 2 6 1 , 10218-10227 Lohman., T. M.. Buialowski. W.. and Overman. L. B. (1988) . . Trends Biochem. Sci'13,-250-255 ' Lucchini, R., Pauli, U., Braun, R., and Koller, T. (1987) J. Mol. Biol. 196,829-843 '

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