Proc. Nadl. Acad. Sci. USA

Vol. 89, pp. 8923-8927, October 1992 Microbiology

Monomers and dimers of the RepA protein in plasmid pSC101 replication: Domains in RepA (DNA replication/DNA-binding proteins/binding sites)

DANIELLE MANEN, LIA-CRISTINA UPEGUI GONZALEZ*, AND LUCIEN CARO Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva 4, Switzerland

Communicated by Werner Arber, June 12, 1992 (received for review March 16, 1992)

ABSTRACT The replication of plasmid pSC101 requires the plasmid-encoded protein RepA. This protein has a double role: it binds to three directly repeated sequences in the pSC101 origin and promotes replication of the plasmid; it binds to two inversely repeated sequences in its promoter region and regulates its own transcription. A series of RepA protein derivatives carrying deletions of the C-terminal region were assayed for specific binding. We found that the last third of the protein is not needed for binding to the various specific sites. Truncated proteins that still bind can also form heterodimers with a wild-type protein. Analysis of band retardation assays conducted with wild-type and truncated proteins indicates that RepA binds to directly repeated sequences as a monomer and to inversely repeated sequences as a dimer.

inversely repeated sequences and autoregulates its transcription in dimeric form.

MATERIALS AND METHODS Bacterial Strains and Plasmids. The bacterial strains used in this study were C600 (supE44 hsdR thi-J thr-J leuB6 lac YJ tonA21) and HB101 [supE44 hsdS20(rBmBi) recA13 ara-14 proA2 LacY) galK2 rpsL20 xyl-5 mtl-i]. Subcloning of different fragments of pSC101 (see below) was done by using plasmid pUC19 (21) or plasmid pBR322 (22). DNA Methodology. Enzymes were purchased from commercial suppliers, and their recommended conditions were employed for the reactions. Cloning procedures, transformations in E. coli, plasmid DNA preparation, and DNA labeling by the Klenow fragment of DNA polymerase were done according to Sambrook et al. (23). Crude Protein Extracts. The proteins were extracted by sonication according to Betermier et al. (24). The quantity of total protein was measured by the method of Bradford (25). Gel Retardation. A 20-plI binding reaction mixture consisted of '15 Ag of protein crude extract, 0.5 pug of calf thymus DNA, 2 Aul of 100 mM EDTA (pH 8), 3 jul of 5x binding buffer [50 mM Tris HCl, pH 8.5/5 mM EDTA/ bovine serum albumin (0.25 mg/ml)/0.5 mM dithiothreitol/25 mM MgCl2/1 M NaCl], and 5 ng of 32P-labeled DNA fragment. Binding was carried out at 16°C for 20 min. The bound complex was analyzed in a 7.5% acrylamide gel. After being run at 150 V for 2 h, the gel was dried and autoradiographed. Plasmid Constructions. The pSC101 coordinates are those used by Churchward et al. (6). For creating deletions at the 3' end, we used existing restriction sites (Hae III at bp 1384 and Sau IIIA at bp 1433) as well as BamHI restriction sites provided by random insertions of an Qt fragment (26) into the repA gene (18). DNA segments starting at the Spe I site (bp 680; Fig. 3) and containing the repA promoter and various portions of the gene were cloned into pUC19, in the direction opposite that of the lac promoter present on this plasmid. Stop codons in all three reading frames were introduced at the 3' end of some of the truncated genes by insertion of the Q fragment. The fragments were called D1 (ending at bp 1028), D2 (bp 1188), D3Q (bp 1335), D3TQ (bp 1384), D3BfQ (bp 1433), D3QfQ (bp 1458), D4Qt (bp 1488), DSM (bp 1602), D6Q (bp 1692), and D7 (bp 1705). The corresponding plasmids are called pUC19-D1, pUC19-D2, pUC19-D3TQ, etc. The plasmids were finally introduced by transformation into C600 cells. Plasmid pUC19-D7-D3BQ1 was constructed by cloning the Pvu II fragment of D3Bf, containing a truncated repA gene followed by the ft fragment, into the HincII site of D7. The resulting plasmid pUCD7-D3BfQ carries both the wild-type and the truncated repA gene, each of them expressed under

The replication of plasmid pSC101 in Escherichia coli depends on the plasmid-encoded protein RepA and on hostencoded proteins such as IHF (1, 2) and DnaA (3, 4). RepA has 316 amino acids and a calculated molecular mass of 37 kDa and is weakly basic with a net charge of +6 (5-9). Interactions between RepA, IHF, and DnaA, binding to the replication origin, are involved in forming structures required for the initiation of replication (10). A 2.2-kilobase HincIIRsa I fragment contains all the functions and sites essential for pSC101 replication including the repA gene (5-8, 11). The replication region (Fig. 1) contains an A+T-rich segment followed by three directly repeated sequences, RS1, RS2, and RS3. The RS1 and RS3 sequences differ by 1 out of 24 base pairs (bp); RS2 is shorter, with 18 homologous base pairs (Fig. 2B). A partially homologous sequence, RS4, is found to the right of RS3; it overlaps the left arm of the palindrome shown as IR1. The repA promoter straddles two palindromic (inversely repeated) sequences, IR1 and IR2, which are also partially homologous to the directly repeated sequences (ref. 12; Figs. 1 and 2B). In vitro the RepA protein appears to bind to all these sequences but more efficiently to the inversely repeated sequences than to the directly repeated sequences (10, 12-14). In vivo it autoregulates the transcription of its mRNA (15-17). Mutations in RepA or within the three repeated sequences upstream can abolish replication (5, 18, 19). This suggests an essential role for the binding of RepA to these sequences in the initiation of replication. The replication of pSC101 has been reviewed recently (20). As a first step in analyzing the functional domains of the protein, deletions of the 3' region of the repA gene were produced. The truncated RepA proteins were assayed for DNA binding. We found that the last third of the C-terminal end is not needed for specific binding to directly repeated or inversely repeated sequences. We also found evidence suggesting that RepA binds to directly repeated sequences and initiates replication in monomeric form but that it binds to The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

*Present address: Institut fur Allgemeine Mikrobiologie, Bern Universitat, Baltzer Strasse 4, 3012 Bern, Switzerland

8923

8924

Microbiology: Manen et al.

Proc. Natl. Acad. Sci. USA 89 (1992) 4F

2(X) luinchl

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FIG. 1. Essential features of the pSC101 origin region. The scale is in base pairs.

the control of a repA promoter. This plasmid was introduced into the recA HB101 strain. We cloned into pUC19 or pBR322 synthetic DNA oligonucleotides, flanked by restriction sites, that were identical with RS1 plus RS2 (pUC-AG), RS3 (pUC-AG3), RS4/IR1 (pUC-SP), and IR2 (pBR-Pr). The DNA fragments used in this work as target sites for the binding of RepA were cut out of these plasmids by using BamHI-EcoRI orBamHI-HindIII (Fig. 2A).

RESULTS Effects of C-Terminal Deletions on the Biding of RepA. We tested the ability of pSC101 RepA proteins with various C-terminal deletions to bind to target DNA sites. A pSC101 fragment containing the repA gene under the control of its own promoter had already been cloned in the high copy number vector pUC19 (plasmid pMX3; ref. 27). The truncated repA genes were cloned similarly (Fig. 3 and Materials and Methods). Crude extracts of proteins from cells harborA RS1+RS2: aattcgagctcggtacccggggatcCAAAMGTCTAGCGGAATTTACAGABGT qctcqaqccatgqqcccctaqGTTTCCATCGCCTTAAATGTCTCCCAGATCGTCTTAAATGTtcqa

ing such plasmids provided enough RepA protein to monitor its binding to the repeated sequences by gel retardation experiments. To detect DNA-binding activity, crude extracts containing intact or truncated RepA were incubated with target DNA fragments. The DNA targets used were either a fragment containing the RS3 repeat sequence, a fragment containing both RS1 and RS2, a fragment named RS4/IR1, containing the sequences between RS3 and IR2, or the inverted repeat sequence IR2. These fragments (described in Materials and Methods and Fig. 2A) were labeled with 32P by filling in the ends with the Klenow fragment of DNA polymerase. The protein-DNA mixtures were loaded on nondenaturing polyacrylamide gels. A summary of the results is shown in Fig. 3. Binding of RepA to the inversely repeated sequences IR2 and IRL. Fig. 4A shows the autoradiogram of a retardation gel monitoring the ability of the truncated RepA proteins to bind to the second inversely repeated sequence IR2 and decrease its mobility in the gel. The fragment was retarded by cell extracts containing the entire protein (D7; lane 10), as well as by extracts containing truncated proteins with deletions of up to 89 amino acids (lanes 5-9). The protein from plasmid D3TH, which lacks the last 105 amino acids (lane 4), and those with longer deletions did not bind to the target site. In

RS3:

AA missing

aattcgagctcggtacccggggatcCAAAG6TCTAGCAAATMTACAGA

gctcgagccatgggcccctagBMTTCCAGATCGTCTTAAATGTCTtcga

RS4/IR1: gatcCCACAACT

6AAGACTMTATTATCA1TTACTAGCCg BGTBTTBAGMCCTTTTCCTGATCATTAATASTAACTGATCG6ctta

IR2:

gatcCATCTCAATTGGTATASTGATTAAAATCACCTAaACCAATTGAGAT~a GTAGAGTTAACCATATCACTAMTTTTMThSTCTSGTTAACTCTACttcga

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TGATTAA

AATTGBTCTAB

GTGATT

FIG. 2. (A) Sequences of the RepA binding target sites used in band-shift experiments. Bold uppercase letters represent pSC101 sequences; lowercase letters represent foreign DNA sequences. Stars indicate the strongly homologous regions shown in Fig. 2B. (B) Directly repeated- and inversely repeated sequences. For the right arms of the inverse repeats, IR1-R and IR2-R, the sequence shown is the reverse complement of the original sequence. Only the central part of the IR2 palindrome is shown. Boxes indicate strongly homologous regions.

Dl D2 D3Q D3m D3BQ D3Q(D40 D5Q

224 170 121 105 89

I

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IR

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-

-

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+

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FIG. 3. Map of the repA gene region, with its promoter, and ofthe various deletion fragments used in this paper. The number of amino acids deleted in each fragment is shown as well as the results of band-shift experiments involving either the directly (RS) or inversely (IR) repeated sequences. A " + " indicates that a retarded band was observed. No attempt was made at quantitating the results. AA, amino acids.

Microbiology: Manen et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

B

A _.l _

mo

-w

s

2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

FIG. 4. Band-shift experiments with the various deletion proteins. Crude extracts from cells containing the indicated plasmid and producing the different proteins indicated in Fig. 2 were incubated with IR2 (A) or with RS3 (B) before they were loaded on the same 7.5% polyacrylamide gel. Lanes: 1, no plasmid; 2, pUC19; 3, pUCD3fl (121 amino acids deleted); 4, pUCD3Tfl (105 amino acids deleted); 5, pUCD3BQ (89 amino acids deleted); 6, pUCD3Qfl (80 amino acids deleted); 7, pUCD4fQ (70 amino acids deleted); 8, pUCDSQ (32 amino acids deleted); 9, pUCD6fl (21 amino acids deleted); 10, pUCD7 (no deletion).

the retarded bands, the mobility of the DNA-RepA complexes increased as the protein fragments became shorter. The segment that we call here RS4/IR1 is composed of the 19-bp RS4 sequence, which is partially homologous to RS, and the 26-bp IR1 palindrome, which overlaps RS4 and extends 15 additional bp to the left of RS4 (Fig. 2A). This region binds RepA in vitro (Fig. 5C, lane 4) and is partially protected by RepA in footprinting experiments (12, 14). The results were similar to those obtained with IR2 (data partially shown; Fig. SC, lanes 3 and 4): the last 89 amino acids were not required for binding. Binding of RepA to the directly repeated sequences RS3 and RSJ plus RS2. Results showing the binding of the RepA fragments to the directly repeated sequences RS3 (Fig. 1) are shown in Fig. 4B. They are similar to those obtained with IR2: the last 89 amino acids are not required for binding. Complexes obtained with the D5Q extract (protein lacking 32 amino acids) and the D6W extract (protein lacking 21 amino acids) did, however, show a double band whose upper component had a mobility that was slightly less than that obtained with the entire RepA (Fig. 4B, lanes 8 and 9). This result has not been explained. The deletions might, in those cases, have affected the folding of the protein or its binding properties, factors that intervene in the mobility of a DNAprotein complex. This phenomenon was not observed with the IR2 probe (Fig. 4A). Similar results were obtained when the DNA segment containing RS1 plus RS2 was used as a probe. The RS2 sequence does not, by itself, bind RepA and, at the concentrations used in our experiments, probably does not bind it

A

B

C

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_

1 2 3 4 5 6

AM

IAW 1

2 3 4 5 6

1 2 3 4 5 6

FIG. 5. Crude protein extracts were incubated with IR2 (A), RS3 (B), or RS4/IR1 (C). Lanes: 1, no plasmid; 2, pUC19; 3, pUCD3BQ (89 amino acids deleted); 4, pUCD7 (wild-type protein); 5, pUCD7D3BQ (wild-type and deleted protein); 6, mixture of pUCD7 and pUCD3Bfl.

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when associated with RS1-that is to say that the mobility behavior of the complex indicated that only one protein was bound to the fragment (data not shown). The DNA targets containing RS3 or IR2 have roughly the same size. Yet their mobility after binding is quite different; the IR2 complex is much slower than the RS3 complex when run on the same gel under identical conditions, whereas the fragments alone have approximately the same mobility (Figs. 4 A and B). It seems likely that this difference is due to a difference in the size or in the amount of protein bound to each target (28). Fig. 2B shows the sequence homologies between the different binding sites. A strong 7-bp homology (GGTCTAG) emerges between them. This suggests that this short consensus sequence might be the primary binding site for the

protein. Does RepA Bind as a Monomer or as a Dimer? In solution the purified RepA protein is mostly in dimeric form (12). We sought to determine whether RepA binds as a monomer or as a dimer to its different specific sites. The shortened RepA derivative encoded by D3Bf still binds to all the sites and, by virtue of its lower molecular mass, its DNA-protein complexes migrate faster in a gel than those made with the wild-type protein (Fig. 5, lanes 3 and 4). We extracted total proteins from HB101 cells harboring the D7-D3BU plasmid. This plasmid (see Materials and Methods) produces two proteins: the intact RepA and the truncated one, both under the control of a repA promoter. After incubation of the crude extract with the inverted sequence IR2, three complexes of different electromobilities were seen on a gel (Fig. 5A, lane 5). Two of them correspond to the complexes formed with the full-length RepA and with the truncated RepA alone. The third band has an intermediate mobility. We interpret this result as indicating that the first two bands are produced by homodimers of either intact RepA or truncated RepA binding to the target DNA and that the third band results from the formation of heterodimers between the two forms of the protein. When two crude extracts containing either the wild-type RepA alone or the truncated RepA alone are mixed, the intermediate band does not appear. Fig. 5C shows similar results, using RS4/IR1 as the target site. The results are quite different when the directly repeated sequences RS3 or RS1 plus RS2 are used as a target: no band of intermediate mobility was detected on the gels. Fig. SB shows the results with RS3 as the target; similar results were obtained with RS1 plus RS2 (data not shown). No difference was noted when, instead ofthe two proteins originating in the same cell, crude extracts containing each of the two proteins alone were mixed. As observed previously, complexes made with IR2 (Fig. 5A, lane 4) migrate much more slowly than those made with RS3 (Fig. SB, lane 4). These experiments also indicate that heterodimers are formed when the protein subunits are synthesized in the same cell but that they cannot be assembled, in appreciable amounts, under our conditions, after extraction. The failure of heterodimers to be formed and bind to the IR sequences when two extracts each containing only one form of the protein are mixed shows that dimerization must precede binding. The results also show that the dimerization domain of the protein does not include the last C-terminal 89 amino acids.

DISCUSSION The data presented here show that 89 amino acids at the C-terminal end of the RepA protein are not needed for binding in vitro to either the directly repeated sequences or the inversely repeated sequences in the origin of replication of plasmid pSC101. Data not presented here have shown that

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Microbiology: Manen et al.

only very short deletions (2-12 amino acids) can be tolerated at the N-terminal end. In vivo replication of the plasmid has more stringent requirements: none of the plasmids with fi insertions, from which the truncated proteins shown in Fig. 3 were derived, were able to replicate (18). A mutation in the third codon ofthe gene alleviates the requirement for the IHF function in replication (29). A point mutation in codon 56 produced the temperature-sensitive mutant pHS1, whereas point mutations in codons 92, 93, and 96 resulted in suppression of that mutation and in a high copy number (9, 27). Thus, the entire protein, or nearly the entire protein, is needed for in vivo replication. In the initial constructions, the truncated proteins carried variable tails of foreign origin, due to adventitious extensions of the open reading frame caused by the construction. We observed that such tails often resulted in erratic binding behavior of the protein. The introduction of an fi fragment, with stop codons in all three reading frames, at the end of each truncated sequence restored coherent binding properties. We found that, with either one of the inversely repeated sequences as a probe, extracts from cells producing both a wild-type RepA protein and a truncated one yielded three bands in a band shift assay: two with mobilities equal to those produced by extracts in which either one of the proteins is present alone and one of intermediate mobility. We interpret this intermediate band as resulting from heterodimers formed between the two proteins. When directly repeated sequences were used as a probe, only two bands, with the mobilities expected for each of the two proteins provided alone, were seen. Our interpretation is reinforced by consideration of the mobility of probes RS3 and IR2 after incubation with an extract containing wild-type RepA. The two probes have nearly the same length (Fig. 2A) and the same mobility (Fig. 4); yet, the protein-bound IR2 migrated much more slowly than the protein-bound RS3. This is consistent with IR2 complexing with a dimer and RS3 with a monomer of RepA. A similar migration behavior for these two probes was observed when purified RepA protein was used (14). Churchward's group has observed that the purified pSC101 RepA protein was present in two forms with different binding properties (G. Churchward, personal communication). They have found that there was no competition for the binding of the purified protein between a DNA fragment containing IR2 and one containing the RS1 direct repeat, although both were binding some protein. This suggests that the purified protein contains two populations, each binding to one target only. We have confirmed these observations with purified RepA, using the segments shown in Fig. 2A (14). Sugiura et al. (12) have found that the purified RepA protein is present mostly in dimeric form. Since we find that the dimeric form is stable and is not assembled efficiently in vitro, we conclude that both the dimeric and the monomeric forms coexist in the cell and in purified protein preparations. Together these data indicate that RepA binds to repeated sequences as a monomer and to inversely repeated sequences as a dimer. They indicate further that dimers bind directly to inversely repeated sequences as opposed to a binding of monomers followed by dimerization. Our data also indicate that approximately one-third of the protein at the C-terminal end and a few amino acids at the N-terminal end are dispensable for dimerization of the RepA protein. The method used here to demonstrate the occurrence of heterodimers had been used previously to determine the heteromeric subunit structure of proteins like GCN4, a yeast transcription factor (30). Wickner et al. (31) have obtained results indicating that the RepA protein of prophage P1 can bind to the repeated sequences of that plasmid only when it is converted from dimers to monomers by the action of two heat shock proteins,

Proc. Natl. Acad. Sci. USA 89 (1992)

first DnaJ and then, in an ATP-dependent reaction, DnaK. They find that the monomer form is stable in dilute solutions. There are some differences between the pSC101 and the P1 situations: in P1 the dimer form does not seem to have an active role and may be involved only in regulating the amount of active, monomeric form present in the cell. In contrast, in pSC101, dimers and monomers seem to have different functions: autoregulation of repA for the dimer and activation of replication for the monomer. Adding ATP did not improve the binding reaction (unpublished results). We find that RepA produced in dnaK mutant cells binds poorly to directly repeated sequences but binds normally to inversely repeated sequences (F. Keppel, personal communication), suggesting, as in the case of P1, a role for DnaK in the dimer-to-monomer transition, but we do not find any effect of dnaJ mutations. We have found that a synthetic DNA fragment containing only the entire IR1 site or one containing the RS4 site, as they were defined by Sugiura et al. (12), do not by themselves bind RepA (data not shown). The RS4/IR1 fragment, which we have used as a probe, and which binds RepA efficiently, extends 19 bp to the left of IR1 and 15 to the left of RS4. The footprint data of Sugiura et al. (12) and our own (14) indicate that protection against DNase I digestion conferred by RepA extends 9 bp to the left of IRL. It must, therefore, be considered that these 9 bp are part of the RepA dimer binding site. Our binding data as well as the competition data mentioned above indicate that RS4 is not a proper binding site for the monomer of RepA and does not seem to have a function of its own. The results presented here indicating that dimers and monomers of the protein are formed in the cell, are stable in vitro, and have different binding properties, as well as the data of Wickner et al. (31), might throw a new light on some previously accepted notions. Thus, it seems now widely accepted that incompatibility, for plasmids of the P1-pSC101 type, is not due to sequestration of the protein (32, 33). The fact, however, that initiation of replication is due not to the entire pool of protein but to a minor form, the regulation of which is poorly understood, could call this belief into question. Various authors (12-14) have demonstrated a stronger binding of pSC101 RepA to inversely repeated sequences than to directly repeated sequences. We, now, might wonder whether this is entirely due to a higher affinity of the protein for these sequences or to the balance of monomer and dimer forms in the extract. In support of the original interpretation, we have found that the protein was more stably bound to inversely repeated sequences than to directly repeated sequences (14). We would like to thank Gordon Churchward for communication of unpublished results; Thomas Goebel, France Keppel, Xia Guixian, Henry Krisch, and Pierre Prentki for helpful discussions; Maryse Pougeon and Dany Rifat for technical assistance; and Edouard Boy de la Tour and Otto Jenni for drawings and photographs. This work was supported by Grant 31.25698.88 from the Swiss National Science Foundation. 1. Gamas, P., Burger, A. C., Churchward, G., Caro, L., Galas, D. & Chandler, M. (1986) Mol. Gen. Genet. 204, 85-89. 2. Stenzel, T. T., Patel, P. & Bastia, D. (1987) Cell 49, 709-717. 3. Hasunuma, K. & Sekiguchi, M. (1977) Mol. Gen. Genet. 154, 225-230. 4. Frey, J., Chandler, M. & Caro, L. (1979) Mol. Gen. Genet. 174, 117-126.

5. Linder, P., Churchward, G. & Caro, L. (1983) J. Mol. Biol. 170, 287-303. 6. Churchward, G., Linder, P. & Caro, L. (1983) Nucleic Acids Res. 11, 5645-5659. 7. Vocke, C. & Bastia, D. (1983) Proc. Nat!. Acad. Sci. USA 80, 6557-6561. 8. Yamaguchi, K. & Yamaguchi, M. (1984) Gene 29, 211-219. 9. Armstrong, K. A., Acosta, R., Ledner, E., Machida, Y.,

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10. 11. 12.

13. 14. 15.

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Pancotto, M., McCormick, M., Ohtsubo, M. H. & Ohtsubo, E. (1984) J. Mol. Biol. 175, 331-348. Stenzel, T. T., MacAllister, T. & Bastia, D. (1991) Genes Dev. 5, 1453-1463. Meacock, P. A. & Cohen, S. N. (1980) Cell 201, 529-542. Sugiura, S., Tanaka, M., Masamune, Y. & Yamaguchi, K. (1990) J. Biochem. 107, 369-376. Vocke, C. & Bastia, D. (1983) Cell 35, 495-502. Xia, G.-X., Manen, D. & Caro, L. (1992) J. Bacteriol., in press. Linder, P., Churchward, G., Xia, G.-X., Yu, Y. Y. & Caro, L. (1985) J. Mol. Biol. 181, 383-393. Vocke, C. & Bastia, D. (1985) Proc. Natl. Acad. Sci. USA 82, 2252-2256. Yamaguchi, K. & Masamune, Y. (1985) Mol. Gen. Genet. 200, 362-367. Manen, D., Izaurralde, E., Churchward, G. & Caro, L. (1989) J. Bacteriol. 171, 6482-6492. Arini, A., Tuscan, M. & Churchward, G. (1991) J. Bacteriol. 174, 456-463. Manen, D. & Caro, L. (1991) Mol. Microbiol. 5, 233-237.

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21. Yanish-Perron, C., Vieira, J. & Messing, J. (1985) Gene 33, 103-119. 22. Bolivar, F. (1978) Gene 4, 121-136. 23. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 24. B6termier, M., Lefrere, V., Koch, C., Alazard, R. & Chandler, M. (1989) Mol. Microbiol. 3, 459-468. 25. Bradford, M. (1976) Anal. Biochem. 72, 248-254. 26. Prentki, P. & Krisch, H. (1984) Gene 29, 303-313. 27. Xia, G.-X., Manen, D., Goebel, T., Linder, P., Churchward, G. & Caro, L. (1991) Mol. Microbiol. 5, 631-640. 28. Hope, I. A. & Struhl, K. (1986) Cell 43, 177-188. 29. Biek, D. & Cohen, S. N. (1989) J. Bacteriol. 171, 2056-2065. 30. Hope, I. A. & Struhl, K. (1987) EMBO J. 6, 2781-2784. 31. Wickner, S. H., Hoskins, J. & McKenney, K. (1991) Proc. Natl. Acad. Sci. USA 88, 7903-7907. 32. Chattoraj, D. K., Mason, R. J. & Wickner, S. H. (1988) Cell 52, 551-557. 33. McEachern, M. J., Bott, M. A., Tooker, P. A. & Helinsky, D. R. (1989) Proc. Natl. Acad. Sci. USA 86, 7942-7946.

Monomers and dimers of the RepA protein in plasmid pSC101 replication: domains in RepA.

The replication of plasmid pSC101 requires the plasmid-encoded protein RepA. This protein has a double role: it binds to three directly repeated seque...
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