THE JOURNAL OF BKILOGICAL CHEMISTRY Vol. 265, No. 6, Issue of February 25, pp. 3464-3466, 1990 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Specificity of RepC Protein in Plasmid pTlS1 DNA Replication*

(Received for publication, August 21, 1989)

Joseph M. ZockS, Patrick Birch, and Saleem A. Khan4 From the DeDartment of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, kennsyluan;a 15261

The plasmid pTl81 of Staphylococcus aureus con- sists of 4437 base pairs and encodes resistance to tet- racycline. Initiation of pTlS1 DNA replication specif- ically requires the plasmid-encoded initiator protein, RepC. The initiator protein binds specifically to a 32- base pair sequence within the pT181 origin of repli- cation. RepC protein also has a nicking-closing activity that is specific for the pTlS1 origin. Replication of pTl81 initiates by covalent extension of the nick and proceeds by a rolling circle mechanism. Two other small, multicopy plasmids pC221 and pS194 belong to the pT181 family and have common structural orga- nization and replication properties. The replication proteins and replication origins of these plasmids have extensive sequence homologies, although they belong to different incompatibility groups. In spite of this homology, the replication proteins and replication origins of these three plasmids do not show any cross- reactivity in vivo. We have carried out a series of in vitro experiments to determine the specificity of pT181-encoded initiator protein, RepC. DNA binding experiments showed that although the binding of RepC to the pT181 origin was very efficient, little or no binding was seen with pC221 and pS194 origins. The nicking-closing activity of RepC was found to be equally efficient with the pC221 and pS194 plasmids. The plasmids pC22 1 and pS 194 replicated efficiently in a Rep&dependent in vitro system. However, repli- cation of these plasmids was greatly reduced in the presence of a competing pT181 origin. The results presented here suggest that nicking-closing by RepC at the origin is not sufficient for maximal replication and that tight binding of RepC to the origin plays an important role in the initiation of DNA replication.

DNA replication is usually regulated at the level of initia- tion. Bacterial plasmids serve as useful systems to study the mechanism and regulation of DNA replication. Replication of most plasmids requires plasmid-encoded initiator proteins in addition to several host proteins (1). Interaction of initiator proteins with origin sequences is a key step in the initiation of DNA replication. The frequency and specificity of this interaction may regulate replication. An important approach towards the elucidation of the mechanism and regulation of

* This work was supported by Grant GM31685 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Eli Lilly and Co., Indianapolis, IN 46285. f Recipient of a National Institutes of Health Research Career

Development Award. To whom correspondence should be addressed. Tel.: 412-648-9025.

DNA replication involves the use of in vitro replication sys- tems and the study of DNA-protein interactions in uitro.

pT181 is a completely sequenced, 4437-base pair (bp)’ plas- mid from Stuphylococcus aureus that encodes tetracycline resistance (2). Genetic and biochemical experiments have shown that a 38-kDa initiator protein (RepC) is required for the initiation of pT181 replication (3, 4). The optimal origin of replication of pT181 consists of 70 bp and also encodes the amino terminal portion of RepC protein (2, 5, 6). RepC has been purified and shown to trigger the in vitro replication of pT181 DNA (7). RepC binds to a 32-bp sequence within the pT181 origin and has origin-specific nicking-closing activities (8, 9). The 3’ OH end generated by the RepC nick serves as the primer for DNA replication by a rolling circle type mech- anism (9).’ The replication of pTl81 both in uiuo and in uitro has been shown to generate single-stranded (SS) DNA and the pT181 origin also acts as the site for termination of replication (10-13). The pT181 replicon also includes two additional regions, pal4 that probably acts as the lagging strand origin and cmp, an enhancer of DNA replication (11, 14).

A number of naturally occurring, small staphylococcal plas- mids, including pT181, pC221, pS194, pC223, and pUB112, share common structural organization (2, 15-17). These five plasmids range in size from 4.2 to 4.6 kilobases and have extensive sequence homologies in their replication regions, although they belong to different incompatibility groups. Three of these plasmids, pT181, pC221, and pS194 were used in the present study. The sequence encoding the Rep proteins and the replication origins of these plasmids have regions of virtual identity as well as regions of divergence (17). Genetic studies have shown that a divergent amino acid sequence near the carboxyl-terminal end of the Rep proteins is involved in their origin recognition specificity (15, 17). The origins of replication of these plasmids have a region of almost perfect homology and a variable region. The constant origin region contains the Rep nick site and can assume a hairpin structure (81.3

In this paper we demonstrate that pC221 and pS194 DNA are efficiently relaxed by RepC protein in the absence as well as presence of competing pT181 DNA. The pC221 and pS194 DNA replicate efficiently in a RepC-dependent in vitro sys- tem, but their replication is greatly reduced in the presence of a competing pT181 origin. Using gel retardation assays we demonstrate that although RepC binds tightly to the pT181 origin, no significant binding of RepC is seen with the pC221 and pS194 origins. These data indicate that the constant origin region is sufficient for RepC-dependent replication, but

1 The abbreviations used are: bp, base pair(s); dNTP, deoxyribo- nucleotide triphosphate; SS, single-stranded plasmid DNA.

* S. Khan, unpublished data. 3 R. Novick, personal communication.

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Specificity of RepC Protein

maximal replication requires the corresponding DNA se- quence in the variable region.

EXPERIMENTAL PROCEDURES

Hactrial Strains--S. aureus strains carrying the pT181, cop-608, pC221, and pS194 plasmids have been described earlier (17). Plasmid pSK41 is a derivative of’pT181 that has a deletion of the cmp region.

DNA Procedures-Plasmid DNA from S. aureus was isolated bv CsCl/ethidium bromide density gradient centrifugation (18). Restric- tion enzymes and the Klenow fragment of DNA polymerase I were purchased from the Bethesda Research Laboratories and used ac- cording to the supplier’s instructions. For the isolation of restriction fragments, various DNAs were treated with the appropriate restric- tion enzymes and the desired fragments were isolated by polyacryl- amide gel electrophoresis followed by crushing and elution (19). Restriction fragments were labeled at their 3’ ends by using [N-“PI dNTPs and the Klenow fragment of Escherichia coli DNA polvmerase . _ I (20).

Purification of RepC Protein and DNA Relaxation Reactions-RepC protein was purified by using an E. coli strain that overproduces this protein as described earlier (7). DNA relaxation reactions (25 ~1) contained 10 mM Tris-Cl, pH 8.0, 100 mM KCl, 10 mM Mg(OAc)p, 10% ethylene glycol, 500 ng of each DNA, and the indicated amount of RepC protein. After incubation at 32 “C for 30 min, reactions were stopped by the addition of 3 ~1 of 50% glycerol, 20 mM EDTA, 0.2% bromphenol blue and electrophoresed on 1% agarose gels using Tris borate-EDTA buffer as described (8). Reaction products were iden- tified by staining the gels with 0.5 pg/ml ethidium bromide and photography under UV light.

In Vitro Replication-Plasmid DNAs were replicated using cell- free extracts from strain RN1786 and purified RepC protein as described earlier (7. 10). Reactions (30 ~1) contained 40 mM Tris-Cl. pH 8.0, 100 mM KCI,’ 12 mM Mg(OAc)P, 1 mM dithiothreitol, 5% ethylene glycol (v/v), 2 mM ATP, 0.5 mM concentration each of UTP, CTP, and GTP, 50 pM dATP, dCTP, dGTP, and dTTP (?‘P at 13,000 cpm/pmol), 50 pM each of NAD and CAMP, 500 pg of crude RN1786 protein extract, 250 ng of template plasmid DNA (unless otherwise indicated), and the indicated amounts of RepC protein. Incubations were carried out at 32 “C for 1 h. DNA was recovered as described earlier (10) and treated with HpaII restriction endonuclease to gen- erate either one (cop-608 and pSK41) or two (pC221 and pS194) fragments. This step was necessary to differentiate cop-608 from pC221 and pS194 DNAs which are similar in size and also to convert various forms of plasmid DNA (supercoiled, nicked open circular, and covalently closed relaxed circular) to one or two bands. The DNA samples were then analyzed by electrophoresis through 1.2% agarose aels (3 V/cm for 16 h) usina Tris acetate EDTA buffer containing 0.5 ., pg/ml ethidium bromide. The gels were dried and subjected to auto- radiography. To measure the extent of replication, the labeled DNA bands were excised from the dried gels and the radioactivity was determined by liquid scintillation counting.

Gel Retardation Assays-The binding of RepC protein to the origins of replication of pT181, pC221, and pS194 was studied by gel mobility shift up assays (21). A nonspecific DNA fragment was used as the negative control. Reactions (20 ~1) contained 10 mM Tris-Cl, pH 8.0, 1 mM dithiothreitol, 1 mM EDTA, 50 mM KCL, 5% glycerol, 0.5% polyvinyl alcohol, 2 Kg of poly(dI-dC), 0.15 ng of “P-labeled restric- tion fragments (specific activity 2.3-3.8 X 10s cpm/pg) and the indicated amount of RepC protein. Incubations were carried out at room temperature for 10 min and the DNA-protein complexes were analyzed by electrophoresis on 5% polyacrylamide gels at 4 “C using Tris borate-EDTA buffer. Gels were run at 10 V/cm for 2.5 h. dried. and subjected to autoradiography.

RESULTS

Comparison of the Replication Origins of pT181, pC221, and pS194-A 70-bp region of pT18I (nucleotides 31-101 on the pTI81 map) contains the functional origin of replication, including the ability to compete with a wild-type origin for its replication (6). A smaller region consisting of about 43 bp (nucleotides 44-86) is sufficient for origin function in the absence of competition (6). The nucleotide sequence of the pT181 origin and the corresponding sequence of the pC221 and pS194 plasmids is shown in Fig. 1. The origin region of these three plasmids can be divided into two regions, the

lnltlation reglon Specificity region

RepC nick RepC binding

t pT181 S'.TTTCTAAAACCGGCT_ACTCTAATAGCCGGTTGGACGCACATACTGTGTGCATATCTGATC.3'

- PC22 1 5'.TTTCTAAAACCGGCTACTCTAATAGCCGGTTaagtGgtaATttTtTtaCCACCCCTCAaC.3'

S'.TTTCTAAAACCGGaTACTCTAATAGCCGGTTaaACcgACATAtTaTGTaCACCCCCGA~C.~' "lg4 60 8-O 7b 8.0 5'0 4b

FIG. 1. Comparison of the replication origins of pT181, pC221, and pS194. The numbering corresponds to the published sequence of pTlS1 (2). Nucleotides in pC221 and pS194 which are identical to pT181 are shown in capitals. The constant origin region of these plasmids is termed the initiation region, whereas the variable region is indicated as the specificity region. Inverted arrows indicate a sequence that can form a hairpin structure (the HpaII stem). The two HpaII sites are present within this inverted repeat sequence. The RepC nick site is indicated by an arrow, and the RepC binding sequence is underlined.

’ 2 3 4 5 6 7 8 9 10 1112

FIG. 2. Binding of RepC protein to the origin sequences. “P- Labeled origin fragments from pT181, pC221, and pS194 were incu- bated with RepC protein, and the DNA-protein complexes were analyzed by polyacrylamide gel electrophoresis as described under “Experimental Procedures.” Lanes 1-3, pT181 origin DNA; lanes 4- 6, pC221 origin DNA; lanes 7-9, pS194 origin DNA; lanes 10-12, nonspecific control DNA. The following amounts of RepC were added to the reactions: lanes I, 4, 7, and 10, none; lanes 2, 5, 8, and II, 170 ng; lanes 3, 6, 9, and 12, 700 ng.

constant region (positions 60-90 in Fig. 1) where all nucleo- tides (except one in the case of pS194) are identical and a variable region (positions 31-59) where only limited homology is seen. The constant region containing two HpaII sites can potentially form a hairpin structure.” The RepC nick site is located in the loop of this hairpin structure (8). Since the replication proteins of pT181 and pC221 only act on their corresponding origins in uiuo (15, 17), the above observations suggest that the variable region of the origin sequence con- tains the recognition specificity of the Rep proteins.

Does RepC Bind to the Origins of Replication of pC221 and pS194?-Earlier results have shown that RepC binds tightly to both the strands of a 32-bp region (nucleotides 37-68 on the pT181 map) within the pT181 origin of replication (9). This binding is presumably involved in the initiation of DNA replication. Since only a part of the RepC binding site (nucle- otides 60-68) is absolutely conserved in pTl81, pC221, and pS194, experiments were carried out to determine if pC221 and pS194 origins bind to RepC protein. The origin-contain- ing 156-bp Mb01 D fragment (pT181), 278-bp TaqI F fragment (pC221), and 287-bp TaqI F fragment (pS194) were checked for their ability to bind to RepC by the gel retardation assays. A lOl-bp nonspecific DNA fragment was used as the control. Although RepC bound efficiently to the pT181 origin DNA, very little or no binding was observed with the pC221 and pS194 origin fragments under the conditions used (Fig. 2). This was not unexpected since pC221 and pS194 contain only

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3486 Specificity of RepC Protein

16 and 22 nucleotides, respectively which are common to the 32-bp RepC binding sequence present in the pT181 origin (Fig. 1). These results suggest that the variable region of the origin plays an important role in RepC binding.

Relaxation of pT181, pC221, and pS194 DNA by RepC Protein-The plasmid cop-608 is a copy mutant of pT181 that contains a origin sequence that is identical to that of pTl81 (2, 22). The cop-608 plasmid was used as the source of the pT181 origin in the present study. We tested the ability of RepC protein to relax the pC221 and pS194 DNA. These DNAs were efficiently relaxed by RepC protein (Fig. 3, lanes 6 and 10). Several other unrelated plasmids were not relaxed by RepC protein (not shown). When decreasing amounts of RepC were used, the relaxation of cop-608, pC221, and pS194 DNAs was reduced to a similar degree (Fig. 3, lanes 3, 7, and 11). We also carried out competition experiments to deter- mine the ability of the pC221 and pS194 DNA to be relaxed by RepC in the presence of a competing pT181 origin (Fig. 3, lanes 13-18). Both pC221 and pS194 DNA competed effec- tively with the wild-type origin and their relaxation by RepC protein was as efficient as that of the cop-608 DNA. Since the RepC nick site is located in the conserved region of the origin sequence, these data indicate that this region is sufficient for relaxation by RepC protein.

In Vitro Replication of pC221 and pS194 DNA in a RepC- dependent System-Plasmids pC221 and pS194 DNA were used as templates in a RepC-dependent in vitro replication system that is specific for the pT181 plasmid (7). The extent of incorporation into cop-608 was about 4-5 pmol of dTMP in the presence of 160 ng of RepC (Fig. 4, lane 2). Plasmids pC221 and pS194 replicated efficiently in the in vitro system (about 65 and 54%, respectively, of the level of cop-608 rep- lication) (Fig. 4, lanes 4 and 9). However, in the presence of competing cop-608 DNA, the replication efficiency of pC221 and pS194 was greatly reduced (10 and 13%, respectively, as compared with cop-608) (Fig. 4, lanes 5 and 10). When a greater amount of RepC (500 ng) was used, both pC221 and pS194 DNA replicated to a greater extent (47 and 49%, respectively, as compared with cop-608) (Fig. 4, lanes 6 and 11). A similar pattern was observed when the amount of the template DNA was reduced in competition experiments (50 ng of each DNA as compared to 250 ng each), thus creating a condition of relative RepC excess (Fig. 4, lanes 7 and 12). In these cases the pC221 and pS194 DNAs replicated to an efficiency of about 63% as compared to the cop-608 DNA which is comparable to their replication in the absence of any competitor DNA. The unrelated pC194 plasmid lacking a pT181 origin did not replicate in the RepC-dependent in vitro system (Fig. 4, lanes 13 and 14). The above results demon- strate that pC221 and pS194 replicate efficiently (although not as well as cop-608) in the absence of any competitor DNA

1 2 3 4 5 6 7 8 9 101112 131415 1617 18

and also under the conditions of RepC excess in the presence of competitor DNA. However, they compete poorly with the pT181 origin when lower concentrations of RepC are used. The replication of cop-608 DNA in vitro has been shown to

I generate single-stranded DNA (10). As observed earlier, the amount of SS DNA obtained during cop-608 replication in- creased as the amount of added RepC was increased (Fig. 4). A small amount of SS pC221 and pS194 DNA can also be seen in reactions that contained 500 ng of RepC protein.

An enhancer of DNA replication, cmp, is located within a 156-bp region between positions 1137 and 1293 on the pT181 map (14). Although plasmids lacking the cmp region have

RePC (W) 0 200 50 15 0 200 50 15 0 200 50 15 200 50 15 200 50 15 normal copy numbers in vivo, they are unstable and have

FIG. 3. Relaxation of DNA by RepC protein. Various super- reduced copy numbers in the presence of a competing plasmid

coiled plasmid DNAs (500 ng each) were incubated with the indicated carrying both the pT181 origin and the cmp regions (14). No amount of RepC protein. Lanes 1-4, cop-608 DNA; lanes 5-8, pC221 such region has yet been identified in the pC221 and pS194 DNA; lunes 9-12, pS194 DNA; lanes 13-15, cop-608 + pC221 DNA; plasmids. We wished to determine if a plasmid carrying a lanes 16-18, cop-608 + pS194 DNA. pT181 origin but lacking the cmp region would compete

SS+

1234567 8 9 10 11 12 13 14

RePC (VJ) 0 160 0 160 160 500 500 0 160 160 500 500160500

FIG. 4. In vitro replication of plasmid DNA. Various plasmid DNAs were replicated in the presence of the indicated amount of RepC protein and analyzed as described under “Experimental Pro- cedures.” Before electrophoresis, the DNAs were cleaved with HpaII restriction endonuclease to generate either one (cop-608) or two (pC221 and pS194) fragments. Lanes I and 2, cop-608 DNA; lanes 3 and 4, pC221 DNA; lanes 5-7, cop-608 + pC221 DNA; lanes 8 and 9, pS194 DNA; lanes 10-12, cop-608 + pS194 DNA; lanes 13 and 14, PC194 DNA. All lanes contained 250 ng each of the indicated DNA, except lanes 7 and 12, which contained 50 ng of each DNA. SS indicates the position of single-stranded plasmid DNA.

12 34

pSK41-r

&b

FIG. 5. In vitro replication of pSK41 DNA. Replication ex- periments were carried out using 500 ng of each DNA. Lanes I and 2, cop-608 DNA; lane 3, pSK41 DNA; lane 4, cop-608 + pSK41 DNA. Lane 1 contained no ReoC. whereas lanes 2-4 contained 160 ne: of RepC.

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Specificity of RepC Protein 3487

effectively with the cop-608 plasmid during in vitro replica- tion. Plasmid pSK41 (3276 bp) was generated by the ligation of the pT181 DcleI A (2772 bp) and DdeI C (504 bp) fragments (2). This plasmid contains the pT181 origin but lacks the cmp region which is contained in the DdeI B fragment (positions 1027-1930 on the pT181 map) (2). Plasmid pSK41 replicated as efficiently as cop-608 in the in vitro system (Fig. 5). Furthermore, the extent of replication of pSK41 DNA was similar to that of cop-608 when both the plasmids were replicated together. These results indicate that the cmp region is not necessary for efficient DNA replication in vitro.

DISCUSSION

The plasmids pT181, pC221, and pS194 of S. aweuS have similar replication regions and structural organization (2,15- 17). The initiator proteins of pT181 and pC221 (RepC and RepD, respectively) have been isolated and shown to initiate replication in uitro by creating a nick within the corresponding origin sequence (8).4 The initiator proteins of pC221 and pS194 have 82 and 75% homologies, respectively, with the amino acid sequence of RepC protein (17). The regions of divergence between these proteins include 20 amino acids at the amino-terminal end, which are probably dispensable for Rep activity (17), the amino acids encoded by the specificity region of the origin (Fig. l), and a 45 amino acid stretch near the carboxyl-terminal end (amino acids 238-282 of RepC) which recognizes the corresponding specific origin (15-17). Earlier experiments showed that the initiator proteins of pT181 and pC221 have strict in uiuo specificity for the corre- sponding origin (15, 17). However, while this manuscript was in preparation the results obtained by Iordanescu showed that if RepC is overproduced in uiuo, it can initiate replication from the pC221 origin in the absence of a competing pT181 origin (23). Similarly, when overproduced, RepD was shown to partially complement the pT181 origin.

The results presented here demonstrate that the nicking- closing of the pC221 and pS194 origins by RepC is as efficient as that of the pT181 origin (Fig. 3). pC221 and pS194 DNA were also relaxed equally well in competition experiments with cop-608 DNA. It has been shown that a 16-nucleotide- long synthetic oligonucleotide (positions 64-79) is sufficient for nicking by RepC when present in a SS form (24). The constant region of the origin (nucleotides 60-83) can assume a hairpin structure (the HpaII stem) both in uiuo and in uitro.3 It is likely that the HpaII hairpin structure (and its DNA sequence) is sufficient for relaxation by RepC protein. Fur- thermore, binding of RepC to the origin variable region con- taining the remaining RepC binding sequence (nucleotides 37-59) is unlikely to be a requirement for its nicking-closing activity. Plasmid pS194 which has a single nucleotide differ- ence in the HpaII stem is expected to have a less stable hairpin structure (Fig. 1). However, pS194 DNA was relaxed as well as the pC221 and cop-608 DNAs. The reasons for this are not clear at present.

Results of RepC-dependent in vitro replication experiments showed that pC221 and pS194 DNA replicated to a consider- able extent, although not as well as the cop-608 DNA (Fig. 4). It is likely that the pC221 and pS194 DNA replicate to a lower degree due to the inability of RepC to bind tightly to the origin variable sequences of these plasmids. These results suggest that although the binding of RepC to its recognition sequence in the variable region may not be necessary for replication, maximal replication requires binding of RepC to this sequence. In the presence of a competing pT181 origin,

’ C. Thomas and W. Shaw, personal communication.

replication of pC221 and pS194 was greatly reduced (Fig. 4). Since the pC221 and pS194 DNAs are relaxed as efficiently as the cop-608 DNA, these results suggest that nicking-closing by RepC is not sufficient for efficient DNA replication. As RepC is usually present in excess during in vitro replication, it may bypass binding to the variable region of the origin and initiate replication of pC221 and pS194 DNA by binding and nicking at the HpuII hairpin. However, in the presence of a competing pT181 origin, the replication of pC221 and pS194 would be greatly reduced due to the specific binding of RepC to the pT181 origin. Since only about 5% of the input template DNA molecules replicate in this in uitro system (lo), a large proportion of the added RepC protein may bind to the pT181 origin, including the nonreplicating molecules. This hypoth- esis is further supported by the observation that when the amount of added RepC was increased, pC221 and pS194 DNAs replicated to a greater extent in the presence of the pT181 origin. Recent in uiuo results have shown that when RepC is present in excess it can drive the replication of the pC221 plasmid in the absence of a competing pT181 origin (23). The in vitro results are generally in agreement with those obtained in uiuo except that the requirements for replication are much more stringent in uiuo. Similar differences in the requirements for in uiuo and in vitro replication were seen in our earlier studies with DNA carrying point mutations in the pT181 origin (6). It is also clear from the present studies that the sequence requirements for in vitro replication are much more stringent than those for nicking-closing by RepC protein.

An enhancer of DNA replication, cmp, has been identified in the pT181 replicon (14). Although cmp is apparently not essential for pT181 replication in uiuo, its presence is neces- sary for replication from the pT181 origin in the presence of a competing pT181 plasmid that carries the cmp region (14). It has been postulated that cmp acts by increasing the effi- ciency of binding between RepC and the pT181 origin. The pSK41 plasmid which is deleted in the cmp region was found to replicate efficiently in uitro in the absence as well as presence of the cop-608 plasmid which contains an intact cmp region (Fig. 5). These data suggest that the cmp region is not required for replication in vitro under the conditions tested. These results also indicate that the inefficient replication of pC221 and pS194 DNA in the presence of a competing pT181 origin is not due to the absence of a cmp element in these plasmids.

Acknowledgments-We thank R. Mahmood, L. Dempsey, and F. Sverdrup for helpful discussions.

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