Proc. Natl. Acad. Sci. USA
Vol. 76, No. 3, pp. 1150-1154, March 1979 Biochemistry
Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K (direct repeats/autogenous regulation/initiation of replication/plasmid-encoded protein)
DAVID M. STALKER, ROBERTO KOLTER, AND DONALD R. HELINSKI Department of Biology, B-022, University of California at San Diego, La Jolla, California 92093
Communicated by Charles Yanofsky, December 14, 1978
ABSTRACT A 2.1-kilobase segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichia cofl. This segment contains a functional origin of replication and a structural gene for a protein, designated r, that is required for the initiation of R6K replication. The nucleotide sequence of a 520-base-pair portion of this 2.1-kilobase segment that includes the functional origin of replication and the region adjacent to the start of the r structural gene was determined. A striking feature of the sequence is the presence of seven 22-base-pair direct repeats joined in tandem in the region adjacent to the start of the 7K gene. A possible role of the tandem repeats in the regulation of expression of the wr protein and the control of initiation of replication of the plasmid R6K is discussed.
coli DNA polymerase I was purchased from Boehringer Mannheim. Amersham was the source of [,y-32P]ATP and [a-32P]dNTPs. Plasmid pRK526 was maintained in the E. coli strain C600trpE5 ( C reactions, hydrazine plus 5.6 M NaCl for C reactions, and hydrazine for C+T reactions. Samples for each reaction contained 10-20 ,ug of unlabeled calf thymus DNA as carrier. Each sample was divided into two portions, and the reactions were carried out for 4 and 30 min. These portions were then recombined before cleavages. Cleavage reactions were carried out in 8% redistilled piperidine for 30 min at 90°C. The samples were dried under reduced pressure, wetted with water to remove residual piperdine, redried, and dissolved in 99% formamide containing 0.05% xylene cyanol and bromphenol blue. Polyacrylamidegels(36 X 26 X 0.04cm) were25%, 12%,or8%, depending on the depth of the sequence destred from the respective end label. Autoradiographs were obtained after placing the gels at -700C with Ilford fast tungstate intensifying screens (Litton).
R6K is a multicopy (10-15 copies per chromosome) plasmid that is 38 kilobases (kb) in size and specifies resistance to the antibiotics ampicillin and streptomycin (1). A physical and genetic map of plasmid R6K has been obtained that includes two bidirectional origins of replication (2-4) and an asymmetric terminus of replication (2, 4). Construction of low molecular weight derivatives of R6K has defined a 2.1-kb segment of this plasmid that contains sufficient information for autonomous replication in Escherichia coli (see Fig. 1) (5). This segment contains an origin of replication and a region (pir) that codes for a protein (designated ir) that is required for initiation of R6K DNA replication (6-8). The pir region is included in two adjacent HindIII fragments (designated 15 and 9) in this 2.1-kb segment of R6K. The origin of replication is located in the region of the junction of the HindIII fragment 9 and the adjacent HindIII fragment 4. HindIII fragments 15 and 9 do not in themselves constitute a functional replicon, but when these two fragments are joined to a CoIEl replicon or to a X phage derivative integrated in the E. coli chromosome, they will support in trans the replication of origin-containing derivatives of R6K (8). These origin-containing derivatives have in common a 380-base-pair (bp) region of R6K that includes the junction of HindIII fragments 9 and 4 (Fig. 1). The nucleotide sequence of a portion of one of these origin-containing derivatives, pRK526, is described in this study. This nucleotide sequence includes a functional origin of replication of R6K and the region adjacent to the start of the structural gene for the 7r protein. MATERIALS AND METHODS Enzymes and DNA Preparations. The restriction endonucleases HindIII, Hae II, and Hae III were purified by the procedure of Greene et al. (9). Bgl II was the generous gift of C. Yanofsky. Bacterial alkaline phosphatase and T4 polynucleotide kinase were from Worthington and P-L Biochemicals, respectively. The Klenow fragment ("large fragment") of E. 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.
Abbreviations: kb, kilobase; bp, base pairs. 1150
Biochemistry: 4
-
Stalker et al.
pir
Proc. Natl. Acad. Sci. USA 76 (1979)
4,'
9
15
B
A
on
_ ¢~~~~~~~~~~~~~~~~~~~~~~~~~~~~
_
680
RESULTS HindII fragments 9 and 15 of plasmid R6K, when present irn E. coli cells, will support the replication of origin-containin~ 9 derivatives of R6K (8). One of these low molecular weight deIIrivatives, plasmid pRK526, consists of a 1300-bp Hae II frag ment containing a functional R6K replication origin joined t 0 a 1400-bp Hae II fragment that specifies resistance to kan 1amycin (Fig. 2) (8). The origin-containing Hae II fragmentit includes portions of R6K HindIII fragments 9 and 4 and thee HindIII site between these fragments. Interruption of thi HindIII site results in a loss of function as a replication origin 1. To determine the sequence of this essential region, we32p end-labeled the HindIII and Bgl II restriction endonucleasee sites of the fragment shown in Fig. 2 with polynucleotide kinasee (5' end) or Klenow DNA polymerase (3' end) at the 4-bp 5'r-
Hae 1I
Bgl 1I
Hae II
111
-Hae
i
II
I I
g~~tHind II
111
_
340
FIG. 3. Gel electrophoresis separation of end-labeled restriction enzyme fragments. Plasmid pRK526 DNA was cut with HindIII or Bgl II, end-labeled, and then digested with Hae III and Hae II, respectively. Fragments were separated on 6% acrylamide slab gels at 80 V until the bromphenol blue dye reached the bottom. Autoradiographs were obtained by 5-min exposures to Kodak x-ray film and the labeled fragments were removed. Gel A shows fragments labeled at the HindIII end. Fragments 680 and 115 are those encompassing the 9*/4* junction. Gel B shows the two labeled DNA fragments surrounding the Bgl II site. (Inset) Gel was stained with 0.4 ltg of ethidium bromide per ml to identify all of the bands produced. Gel a represents pRK526 DNA cleaved with HindIII and Hae II (a 25-bp labeled fragment produced by the cleavage runs off the gel under these conditions). Gel b shows pRK526 cleaved with both Bgl II and Hae II. The labeled fragments are indicated by arrows and correspond to the bands in A and B. Either 3'- or 5'-end-labeled DNAs will generate the same labeled bands.
4*
Hindil
J4ae I
_N
SM1 940
A_
FIG. 1. Map of the region containing a functional origin of replication for plasmid R6K. 2, 4, 9, and 15 refer to HindIII fragments on the R6K physical map (5). The region enclosed by the dashed lines represents the 2.1-kb segment that carries sufficient information for replication as a plasmid. The asymmetric terminus for replication is designated ter. The functional origin of replication is designated ori and the pir gene codes for the ir protein.
9*
1151
Kan
FIG. 2. A partial restriction map of plasmid pRK526 and the 1300-bp Hae II fragment containing the origin of replication. 9* and 4* denote portions of R6K HindIII fragments 9 and 4. The heavy bar shows the region of DNA whose sequence was determined. Solid arrows indicate sequences obtained by 3'-end labeling of the Bgl II and HindIII ends; dashed arrows are sequences obtained by 5'-end labeling. Hae III restriction enzyme sites are indicated as III. Kan refers to the 1400-bp fragment specifying kanamycin resistance and its location on pRK526. A detailed physical map of this plasmid is presented elsewhere (8).
overhangs generated by these restriction enzymes (15, 16). The directionality of sequence determination from each end label and the total region whose sequence was determined are also shown in Fig. 2. The DNA fragments isolated after end labeling of the Bgl II and HindIII restriction endonuclease sites are shown in Fig. 3. DNA end labeling at the two HindIII enzyme sites (one located in the Hae II kanamycin fragment) and digestion with Hae III generates nine bands with four labeled fragments of 680, 500, 115, and 25 bp, respectively. The fragments that are 680 bp and 115 bp long are those encompassing the 9/4 HindIII junction. pRK526 DNA cleaved and labeled at the Bgl II site and then digested with Hae II gives three separate fragments when fractioned on 6% polyacrylamide gels. Two of these (940 bp and 340 bp) are labeled at the Bgl II site.
Chemical degradations were carried out on the four labeled DNA fragments described above; selected gels from the 520-bp sequence are displayed in Fig. 4. Gels a-e represent the sequences labeled at the 3' end of the HindIII site, while autoradiograms f-i represent sequences labeled at the 5' end of the Bgl II site. Regions that are of particular interest include a run of eight adenine residues bound by two guanine residues shown
1152
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Stalker et al. a
d
c
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I
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FIG. 4. Representative sequencing gels prepared from the end-labeled restriction enzyme fragments. Gels a, b, f, and g are 25% acrylamide gels; the others are 12% gels. Gels a-e contain sequences labeled at the 3' end of the HindIII site. Gels f-i contain sequences obtained by 5'-end labeling of the Bgl II site. Gel a, bases 1 through -28; gel b, bases 2 through 30; gel c, bases -21 through -58; gel d, bases 22 through 61; and gel e, bases 80 through 95. Brackets indicate the C-T-C-T-C residues of the first three direct repeats. Gel f, bases 277 through 252; gel g, bases 278 through 302; gel h, bases 307 through 339; and gel i, bases 249 through 217.
in gel d, three tandem direct repeats indicated by brackets in gel e; and a gap in the sequence (position -33) observed in gel c. Gaps in DNA sequencing gels have been described by
Ohmori et al. (17) as possible methylated cytosine residues due to the inability of hydrazine to chemically react with these modified bases. Sequence analysis of the complementary strand (data not shown) has revealed a guanine residue at the position complementary to the missing base. A gap was also detected in the sequence of this complementary strand (position -35) with a guanine residue residing at the opposite position. It is
likely, therefore, that methylated cytosines occupy these two positions.
The entire 520-bp sequence is displayed in Fig. 5, with each base confirmed by sequence analysis of the complementary strand. The base pairs are numbered in both directions from the HindIII site. Positions 12 through 90 constitute a region that is 80% A-T rich, a feature common to several replication origins. A possible RNA polymerase recognition and binding site (18, 19) that contains limited 2-fold rotational symmetry is located between positions 49 and 87. All reading frames for translation
Biochemistry:
Stalker et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
1153
HinfI THhaI HinfI 5' GAGAGTCAATTTTATCTCATGTTTTAGCAACGTACTATCCACTCATG-TTTAACGCTTTTATGAflGTC~GCTATATTACCCCAAACCCGCGECATGCTTGGCAAAGAT
31 CTCTCAGTTAAAATAGAGTACAAAATCGTTGCATGATAGGTGAGTACAAATTGCGAAAATACTCAGCGATATAATGGGGTTTGGGCGCGGTACGAACCGTTTCTA 400
360
380
320
340
MboI AZuI
zII MboI I
A ZuI
GATTAAAAAGCCACCTGTTTTAAGCTAGATCTGAAGATCAGCAGTTCAACCTGTTGATAGTACGTACTAAGCTCTCATGTTTCACGTACTAAGCTCTCATGTTTA CTAATTTTTCGGTGGACAAAATTCGATCTAGACT-TCTAGTCGTCAAGTTGGACAACTATCATGCATGATTCGAGAGTACAAAGTGCATGATTCGAGAGTACAAAT 300
280
2 60
WUAI
AZUI
24 0 AZuI
22 0 AZuI
AZuI
ACGTACTAAGCTCTCATGTTTAACGAACTAAACCCTCATGGCTAACGTACTAAGCTCTCATGGCTAACGTACTAAGCTCTCATGTTTCACGTACTAAGCTCTCAT TGCATGATTCGAGAGTACAAATTGCTTGATTTGGGAGTACCGATTGCATGATTCGAGAGTACCGATTGCATGATTCGAGAGTACAAAGTGCATGATTCGAGAGTA 200
180
160
140
120
100
AZI
GTTTGAAC* CAAACTTG
AAA
hfr~~~~~~~tLL ~~~~~~HindIII
TAGCCTCTA AGGTTTTAAGTTTTATAAGAAAAAAAAGAATATATAAGGCTTTTAAAGCTTTTAAGGT AiATAT*ATCAGCAACTT4 TTATATJTAGTCGTTGAAT TATCGGAGAT CAAAATTCAAAATATTCTTTTTTTTCTTATATATTCCGAAAATTTCGAAAATTCCA
TUATTTA80
60
40
20
1
EaoRII HaeII MZI TTAACGGTTGTGGACAACAAGCCAGGGATGTAACGCACTGAGAAGCCCTTAGAGCT-TCAAAGCAATTTTCAGTGACACAGGAACACTTAACGGCTGACA .... 3' ATACAT TGCTGACTCTTCGGGAATCTCGGAGAGTTTCGTTAAAAGTCACTGTGTCCTTGTGAATTGCCGACTGT.... 5 -20 -40 -60 -80 -100 FIG. 5. Nucleotide sequence of the 520-bp segment of plasmid pRK526. Bases are numbered from the HindIII site, and restriction enzyme sites are indicated. Arrows show the 22-bp direct repeats. Regions of 2-fold rotational symmetry are indicated by arrows between strands. The bracketed bases indicate a possible RNA polymerase binding site (promotor); the positions of the tentatively identified methylated cytosine residues are indicated by asterisks.
are interrupted by nonsense codons in both directions, indicating that no large polypeptides are coded for in this region. However, coding for small functional peptides cannot be ruled out. An exception is a possible GUG start at base 370, which is open with respect to translation as far as the sequence has been determined. Seven tandem 22-bp direct repeats are designated by arrows in Fig. 5 starting at position 93 and continuing through 246. An eighth repeat unit in the region 362 to 383 contains the open GUG start. As shown in Fig. 6, the repeat units are nearly-identical in sequence, with the exception of the middle unit which contains five different bases. DISCUSSION The nucleotide sequence of the region containing a functional origin of plasmid R6K replication that is present in pRK526, a low molecular weight derivative of plasmid R6K, has been determined. The replication of pRK526 in E. coli requires the presence of HindIl fragments 9 and 15 of R6K, which code for a protein (Xr) that is required for the initiation of R6K replication (6-8). These fragments can be incorporated into E. coli cells by joining them to a ColEl replicon or to a X phage derivative that is integrated in the host chromosome (8). Plasmid pRK526 is maintained in cells carrying HindIII fragments 9 and 15 at a copy number similar to that of the parent R6K plasmid (10-15 copies per chromosome) (8). The functional origin of replication in pRK526 described in this study is contained within portions of HindIll fragment 9 and the adjacent HindIll fragment 4 (8). The close proximity of a gene speci5'
AACGTACTACDC®CTCATGTTT
3'
TI T (DA C G T A C T A A S C T C T C A TIG I I
QA C G T A C T A A G C T C T C A G T T AACGTACTAAGCTCTCATGTTT A A C GSA C T A A®C®C T C AT GGT A A C G T A C T A A G CT C T C A GZI T AACGTACTAAGCTCTCATGTTT (A C GST A C T A A G C T C T C A TIG T I T
FIG. 6. Nucleotide sequence of the eight 22-bp direct repeats which are read from left to right in Fig. 5. The top repeat unit is separated from the seven tandem repeats and spans bases 362-383. Circled residues indicate differences between bases.
fying a protein required for the initiation of DNA replication to the origin of replication has been shown for several different replicons. Examples of this close spatial relationship are the cisA protein of phage qX174 (20-22), the 0 protein of phage X (23-25), and the T antigen of the animal virus simian virus 40 (26-29). In the case of the R6K derivatives described in this study, evidence has been obtained for the start of translation of the ir protein in HindIII fragment 9 (unpublished observations). The nucleotide sequence obtained for a portion of fragment 9 contains a possible promoter for leftward transcription of the pir gene (Fig. 5). This site contains an RNA polymerase recognition sequence (nucleotide bases 49 through 60) within a region having limited 2-fold rotational symmetry. The sequence -T-A-T-A-T-T-A- at positions 74 through 80 differs by one base from the one described by Pribnow as an RNA polymerase binding site (18). If this is the case, transcription would begin at position 86, proceed through the region containing the direct repeats, and continue through the rest of fragment 9 and all of fragment 15 to include the extra pir structural gene. Binding of the or protein to the direct repeat region (a presumptive operator) could serve to regulate gx synthesis by shutting off transcription. This would provide an autoregulatory mechanism for the regulation of DNA replication and possibly plasmid copy number. The finding of a similar copy number of plasmid derivatives carrying this origin fragment in E. coli cells carrying the pir gene either on the chromosome or on a multicopy plasmid is consistent with an autoregulation of ir protein synthesis (8). Direct repeats function in regulating the expression of the cI and crc genes in phage A (30, 31). The CI and Cro repressor proteins bind to the 17-bp repeats that constitute the left- and right-hand operators and promoters of the phage (32). It has been proposed that this binding serves to autoregulate transcription of these repressor genes (32). Tandem repeats also have recently been observed at or near the origins of DNA replication of phage A (33) and simian virus 40 (29). It is also possible that the direct repeats found near the functional origin of plasmid R6K are involved in regulating other properties of this plasmid such as incompatibility or segregation or both. Initiation of R6K DNA replication is rifampicin sensitive, indicating an involvement of RNA polymerase (6, 7). The fact
1154
Biochemistry: Stalker et al.
that pRK526 is not maintained in E. coli cells if a DNA fragment is inserted in the HindIII site at the 9/4 junction raises the possibility that an origin RNA primer is synthesized across this HindIII site. RNA polymerase conceivably could initiate this RNA primer synthesis in the vicinity of the EcoRII site at nucleotide positions -20 to -34, which contain an RNA polymerase recognition sequence (Fig. 5). The primer would then be synthesized across the 9/4 junction and possibly terminate at the stretch of adenine residues at positions 21-29 (Fig. 5). Adenine residues have been implicated in rho-independent termination of transcription by RNA polymerase (19). Runs of adenine residues of varying lengths (five to nine bases) also have been observed near origins of replication of plasmid ColEl (34, 35), phage X (33), the papovaviruses simian virus 40 (28. 29), polyoma (36), and BK (37), the origin of E. coli replication (38, 39), and the leader sequence of the tryptophan operon (40). The significance of the putative methylated cytosine residues at positions -33 and -35 with regard to the replication origin activity of this region is not known. These modified bases are part of an EcoRII restriction endonuclease cleavage site (41) and are recognized by both the EcoRII and cellular methylase activities (42). Methylated cytosine residues are found in the region of the origin for replication of plasmid CoWEl, but in this case methylation of these bases is not required for maintenance of the plasmid. It is unclear whether the origin fragment of R6K whose sequence we have determined corresponds to either the a or A origins previously mapped by electron microscopy (3, 5). Attempts to isolate other origin-containing fragments from R6K by the transcomplementation system have proven unsuccessful. It is clear, however, that the nucleotide sequence described contains an origin of replication in the region of the HindIII site that is functional in the presence of ir protein. Whether the ir protein recognizes a sequence of nucleotides at this origin or interacts with another polypeptide involved in the initiation event at this sequence remains to be determined. We are deeply indebted to T. Friedman, P. Laporte, and P. Deinenger for introducing us to sequencing techniques and providing facilities for the initial experiments and helpful suggestions. We are grateful to M. Inuzuka and M. Kahn for helpful discussions and critical reading of the manuscript. This work was supported by grants from the National Institute of Allergy and Infectious Diseases (AI-07194) and National Science Foundation (PCM77-06533). D.S. was supported by a U.S. Public Health Service Postdoctoral Fellowship (1-F32GM06542). R.K. is the recipient of a University of California Predoc-
toral Fellowship. 1. Kontomichalou, P., Mitani, M. & Clowes, R. C. (1970) J. Bacteriol. 104, 34-43. 2. Lovett, M. A., Sparks, R. B. & Helinski, D. R. (1975) Proc. Natl. Acad. Sci. USA 72,2905-2909. 3. Crosa, J. H., Luttropp, L. K. & Falkow, S. (1976) J. Bacteriol. 126, 454-466. 4. Kolter, R. & Helinski, D. R. (1978) J. Mol. Biol. 124,428-441. 5. Kolter, R. & Helinski, D. R. (1978) Plasmid 1, 571-580. 6. Inuzuka, M. & Helinski, D. R. (1978) Biochemistry 17, 25672573. 7. Inuzuka, M. & Helinski, D. R. (1978) Proc. Natl. Acad. Sci. USA
75,5381-5385. 8. Kolter, R., Inuzuka, M. & Helinski, D. R. (1978) Cell 15, 1199-1208. 9. Greene, P. J., Heyneker, H. L., Bolivar, F., Rodriquez, R. L., Betlach, M. C., Covarrubias, A. A., Backman, K., Russel, D. J.,
Proc. Natl. Acad. Sci. USA 76 (1979) Tait, R. & Boyer, H. W. (1978) Nucleic Acids Res. 5, 23732380. 10. Bazaral, M. & Helinski, D. R. (1968) J. Mol. Biol. 36, 185194. 11. Murray, K. (1973) Biochem. J. 131,569-576. 12. Richardson, C. C. (1971) in Procedures in Nucleic Acids Research, eds. Cantoni, G. L. & Davis, D. R. (Harper & Row, New York), Vol. 11, pp. 815-828. 13. Klenow, H., Overgaard-Hansen, K. & Patkar, S. A. (1971) Eur. J. Biochem. 22,371-381. 14. Maxam, A. M. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74,560-564. 15. Danna, K. J., Sack, G. H. & Nathans, P. (1973) J. Mol. Biol. 78, 363-376. 16. Pirrotta, V. (1976) Nucleic Acids Res. 3, 1747-1760. 17. Ohmori, H., Tomizawa, J. & Maxam, A. M. (1978) Nucleic Acids Res. 5, 1479-1485. 18. Pribnow, D. (1975) Proc. Natl. Acad. Sci. USA 72, 784-789. 19. Gilbert, W. (1976) in RNA Polymerase, eds. Losick, R. & Chamberlin, M. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 193-203. 20. Baas, P. D., Jansz, H. S. & Sinsheimer, R. L. (1976) J. Mol. Biol. 102,633-656. 21. Eisenberg, S., Griffith, J. & Kornberg, A. (1977) Proc. Natl. Acad. Sci. USA 74,3198-3202. 22. Sanger, F., Air, G. M., Barrell, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., Slocombe, P. M. & Smith, M. (1978) Nature (London) 265,687-695. 23. Ogawa, T. & Tomizawa, J. (1968) J. Mol. Biol. 38,217-225. 24. Schnos, M. & Inman, R. B. (1970) J. Mol. Biol. 51, 61-73. 25. Dove, W. F., Inokuchi, H. & Stevens, W. F. (1971) in The Bacteriophage Lambda, ed. Hershey, A. D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 747-771. 26. Rundell, K., Collins, J. K., Tegtmeyer, P., Ozer, H. L., Lai, C-J. & Nathans, D. (1977) J. Virol. 21, 636-646. 27. Carroll, R. B. & Smith, A. E. (1976) Proc. Natl. Acad. Sci. USA 73,2254-2258. 28. Fiers, W., Contreras, R., Haegeman, G., Rogius, R., Van de Voorde, A., Van Heuverswyn, H., Van Herreweghe, J., Volckaert, G. & Ysebart, M. (1978) Nature (London) 273, 113-120. 29. Reddy, V. B., Thimmappaya, B., Dhar, R., Subramanian, K. N., Zain, B. S., Pan, J., Ghosh, P. K., Celma, M. L. & Weissman, S. M. (1978) Science 200, 494-502. 30. Maniatis, T. & Ptashne, M. (1973) Proc. Natl. Acad. Sci. USA 70, 1531-1535. 31. Folkmanis, A., Takeda, Y., Simuth, J., Gussin, G. & Echols, H. (1976) Proc. Natl. Acad. Sci. USA 73,2249-2253. 32. Ptashne, M., Backman, K., Humayun, M. Z., Jeffery, A., Maurer, R., Meyer, B. & Sauer, R. T. (1976) Science 194, 156-161. 33. Schwarz, E., Scherer, G., Hobom, G. & Kossel, H. (1978) Nature (London) 272, 410-414. 34. Tomizawa, J., Ohmori, H. & Bird, R. E. (1977) Proc. Natl. Acad. Sci. USA 74, 1865-1869. 35. Bastia, D. (1977) Nucleic Acids Res. 4, 3123-3142. 36. Soeda, E., Kimura, G. & Miura, K. (1978) Proc. Natl. Acad. Sci. USA 75, 162-166. 37. Dhar, R., Lai, C-J. & Khoury, G. (1978) Cell 13,345-358. 38. Sugimoto, K., Oka, A., Sugisaki, H., Takanami, M., Nishimura, A., Yasuda, Y. & Hirota, Y. (1979) Proc. Natl. Acad. Sci. USA 76, 575-579. 39. Meijer, M., Beck, E., Hansen, F. G., Bergmans, H. E. N., Messer, W., von Meyenburg, K. & Schaller, H. (1979) Proc. Natl. Acad. Sci. USA 76,580-584. 40. Bertrand, K., Korn, L., Lu, F., Platt, T., Squires, C. L., Squires, C. & Yanofsky, C. (1975) Science 189, 22-25. 41. Bigger, C. H., Murray, K. & Murray, N. E. (1973) Nature (London) New Biol. 244, 7-10. 42. May, M. S. & Hattman, S. (1975) J. Bacteriol. 122, 129-138.
Vol. 76, No. 3, pp. 1150-1154, March 1979 Biochemistry
Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K (direct repeats/autogenous regulation/initiation of replication/plasmid-encoded protein)
DAVID M. STALKER, ROBERTO KOLTER, AND DONALD R. HELINSKI Department of Biology, B-022, University of California at San Diego, La Jolla, California 92093
Communicated by Charles Yanofsky, December 14, 1978
ABSTRACT A 2.1-kilobase segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichia cofl. This segment contains a functional origin of replication and a structural gene for a protein, designated r, that is required for the initiation of R6K replication. The nucleotide sequence of a 520-base-pair portion of this 2.1-kilobase segment that includes the functional origin of replication and the region adjacent to the start of the r structural gene was determined. A striking feature of the sequence is the presence of seven 22-base-pair direct repeats joined in tandem in the region adjacent to the start of the 7K gene. A possible role of the tandem repeats in the regulation of expression of the wr protein and the control of initiation of replication of the plasmid R6K is discussed.
coli DNA polymerase I was purchased from Boehringer Mannheim. Amersham was the source of [,y-32P]ATP and [a-32P]dNTPs. Plasmid pRK526 was maintained in the E. coli strain C600trpE5 ( C reactions, hydrazine plus 5.6 M NaCl for C reactions, and hydrazine for C+T reactions. Samples for each reaction contained 10-20 ,ug of unlabeled calf thymus DNA as carrier. Each sample was divided into two portions, and the reactions were carried out for 4 and 30 min. These portions were then recombined before cleavages. Cleavage reactions were carried out in 8% redistilled piperidine for 30 min at 90°C. The samples were dried under reduced pressure, wetted with water to remove residual piperdine, redried, and dissolved in 99% formamide containing 0.05% xylene cyanol and bromphenol blue. Polyacrylamidegels(36 X 26 X 0.04cm) were25%, 12%,or8%, depending on the depth of the sequence destred from the respective end label. Autoradiographs were obtained after placing the gels at -700C with Ilford fast tungstate intensifying screens (Litton).
R6K is a multicopy (10-15 copies per chromosome) plasmid that is 38 kilobases (kb) in size and specifies resistance to the antibiotics ampicillin and streptomycin (1). A physical and genetic map of plasmid R6K has been obtained that includes two bidirectional origins of replication (2-4) and an asymmetric terminus of replication (2, 4). Construction of low molecular weight derivatives of R6K has defined a 2.1-kb segment of this plasmid that contains sufficient information for autonomous replication in Escherichia coli (see Fig. 1) (5). This segment contains an origin of replication and a region (pir) that codes for a protein (designated ir) that is required for initiation of R6K DNA replication (6-8). The pir region is included in two adjacent HindIII fragments (designated 15 and 9) in this 2.1-kb segment of R6K. The origin of replication is located in the region of the junction of the HindIII fragment 9 and the adjacent HindIII fragment 4. HindIII fragments 15 and 9 do not in themselves constitute a functional replicon, but when these two fragments are joined to a CoIEl replicon or to a X phage derivative integrated in the E. coli chromosome, they will support in trans the replication of origin-containing derivatives of R6K (8). These origin-containing derivatives have in common a 380-base-pair (bp) region of R6K that includes the junction of HindIII fragments 9 and 4 (Fig. 1). The nucleotide sequence of a portion of one of these origin-containing derivatives, pRK526, is described in this study. This nucleotide sequence includes a functional origin of replication of R6K and the region adjacent to the start of the structural gene for the 7r protein. MATERIALS AND METHODS Enzymes and DNA Preparations. The restriction endonucleases HindIII, Hae II, and Hae III were purified by the procedure of Greene et al. (9). Bgl II was the generous gift of C. Yanofsky. Bacterial alkaline phosphatase and T4 polynucleotide kinase were from Worthington and P-L Biochemicals, respectively. The Klenow fragment ("large fragment") of E. 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.
Abbreviations: kb, kilobase; bp, base pairs. 1150
Biochemistry: 4
-
Stalker et al.
pir
Proc. Natl. Acad. Sci. USA 76 (1979)
4,'
9
15
B
A
on
_ ¢~~~~~~~~~~~~~~~~~~~~~~~~~~~~
_
680
RESULTS HindII fragments 9 and 15 of plasmid R6K, when present irn E. coli cells, will support the replication of origin-containin~ 9 derivatives of R6K (8). One of these low molecular weight deIIrivatives, plasmid pRK526, consists of a 1300-bp Hae II frag ment containing a functional R6K replication origin joined t 0 a 1400-bp Hae II fragment that specifies resistance to kan 1amycin (Fig. 2) (8). The origin-containing Hae II fragmentit includes portions of R6K HindIII fragments 9 and 4 and thee HindIII site between these fragments. Interruption of thi HindIII site results in a loss of function as a replication origin 1. To determine the sequence of this essential region, we32p end-labeled the HindIII and Bgl II restriction endonucleasee sites of the fragment shown in Fig. 2 with polynucleotide kinasee (5' end) or Klenow DNA polymerase (3' end) at the 4-bp 5'r-
Hae 1I
Bgl 1I
Hae II
111
-Hae
i
II
I I
g~~tHind II
111
_
340
FIG. 3. Gel electrophoresis separation of end-labeled restriction enzyme fragments. Plasmid pRK526 DNA was cut with HindIII or Bgl II, end-labeled, and then digested with Hae III and Hae II, respectively. Fragments were separated on 6% acrylamide slab gels at 80 V until the bromphenol blue dye reached the bottom. Autoradiographs were obtained by 5-min exposures to Kodak x-ray film and the labeled fragments were removed. Gel A shows fragments labeled at the HindIII end. Fragments 680 and 115 are those encompassing the 9*/4* junction. Gel B shows the two labeled DNA fragments surrounding the Bgl II site. (Inset) Gel was stained with 0.4 ltg of ethidium bromide per ml to identify all of the bands produced. Gel a represents pRK526 DNA cleaved with HindIII and Hae II (a 25-bp labeled fragment produced by the cleavage runs off the gel under these conditions). Gel b shows pRK526 cleaved with both Bgl II and Hae II. The labeled fragments are indicated by arrows and correspond to the bands in A and B. Either 3'- or 5'-end-labeled DNAs will generate the same labeled bands.
4*
Hindil
J4ae I
_N
SM1 940
A_
FIG. 1. Map of the region containing a functional origin of replication for plasmid R6K. 2, 4, 9, and 15 refer to HindIII fragments on the R6K physical map (5). The region enclosed by the dashed lines represents the 2.1-kb segment that carries sufficient information for replication as a plasmid. The asymmetric terminus for replication is designated ter. The functional origin of replication is designated ori and the pir gene codes for the ir protein.
9*
1151
Kan
FIG. 2. A partial restriction map of plasmid pRK526 and the 1300-bp Hae II fragment containing the origin of replication. 9* and 4* denote portions of R6K HindIII fragments 9 and 4. The heavy bar shows the region of DNA whose sequence was determined. Solid arrows indicate sequences obtained by 3'-end labeling of the Bgl II and HindIII ends; dashed arrows are sequences obtained by 5'-end labeling. Hae III restriction enzyme sites are indicated as III. Kan refers to the 1400-bp fragment specifying kanamycin resistance and its location on pRK526. A detailed physical map of this plasmid is presented elsewhere (8).
overhangs generated by these restriction enzymes (15, 16). The directionality of sequence determination from each end label and the total region whose sequence was determined are also shown in Fig. 2. The DNA fragments isolated after end labeling of the Bgl II and HindIII restriction endonuclease sites are shown in Fig. 3. DNA end labeling at the two HindIII enzyme sites (one located in the Hae II kanamycin fragment) and digestion with Hae III generates nine bands with four labeled fragments of 680, 500, 115, and 25 bp, respectively. The fragments that are 680 bp and 115 bp long are those encompassing the 9/4 HindIII junction. pRK526 DNA cleaved and labeled at the Bgl II site and then digested with Hae II gives three separate fragments when fractioned on 6% polyacrylamide gels. Two of these (940 bp and 340 bp) are labeled at the Bgl II site.
Chemical degradations were carried out on the four labeled DNA fragments described above; selected gels from the 520-bp sequence are displayed in Fig. 4. Gels a-e represent the sequences labeled at the 3' end of the HindIII site, while autoradiograms f-i represent sequences labeled at the 5' end of the Bgl II site. Regions that are of particular interest include a run of eight adenine residues bound by two guanine residues shown
1152
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Stalker et al. a
d
c
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I
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An:
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:
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FIG. 4. Representative sequencing gels prepared from the end-labeled restriction enzyme fragments. Gels a, b, f, and g are 25% acrylamide gels; the others are 12% gels. Gels a-e contain sequences labeled at the 3' end of the HindIII site. Gels f-i contain sequences obtained by 5'-end labeling of the Bgl II site. Gel a, bases 1 through -28; gel b, bases 2 through 30; gel c, bases -21 through -58; gel d, bases 22 through 61; and gel e, bases 80 through 95. Brackets indicate the C-T-C-T-C residues of the first three direct repeats. Gel f, bases 277 through 252; gel g, bases 278 through 302; gel h, bases 307 through 339; and gel i, bases 249 through 217.
in gel d, three tandem direct repeats indicated by brackets in gel e; and a gap in the sequence (position -33) observed in gel c. Gaps in DNA sequencing gels have been described by
Ohmori et al. (17) as possible methylated cytosine residues due to the inability of hydrazine to chemically react with these modified bases. Sequence analysis of the complementary strand (data not shown) has revealed a guanine residue at the position complementary to the missing base. A gap was also detected in the sequence of this complementary strand (position -35) with a guanine residue residing at the opposite position. It is
likely, therefore, that methylated cytosines occupy these two positions.
The entire 520-bp sequence is displayed in Fig. 5, with each base confirmed by sequence analysis of the complementary strand. The base pairs are numbered in both directions from the HindIII site. Positions 12 through 90 constitute a region that is 80% A-T rich, a feature common to several replication origins. A possible RNA polymerase recognition and binding site (18, 19) that contains limited 2-fold rotational symmetry is located between positions 49 and 87. All reading frames for translation
Biochemistry:
Stalker et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
1153
HinfI THhaI HinfI 5' GAGAGTCAATTTTATCTCATGTTTTAGCAACGTACTATCCACTCATG-TTTAACGCTTTTATGAflGTC~GCTATATTACCCCAAACCCGCGECATGCTTGGCAAAGAT
31 CTCTCAGTTAAAATAGAGTACAAAATCGTTGCATGATAGGTGAGTACAAATTGCGAAAATACTCAGCGATATAATGGGGTTTGGGCGCGGTACGAACCGTTTCTA 400
360
380
320
340
MboI AZuI
zII MboI I
A ZuI
GATTAAAAAGCCACCTGTTTTAAGCTAGATCTGAAGATCAGCAGTTCAACCTGTTGATAGTACGTACTAAGCTCTCATGTTTCACGTACTAAGCTCTCATGTTTA CTAATTTTTCGGTGGACAAAATTCGATCTAGACT-TCTAGTCGTCAAGTTGGACAACTATCATGCATGATTCGAGAGTACAAAGTGCATGATTCGAGAGTACAAAT 300
280
2 60
WUAI
AZUI
24 0 AZuI
22 0 AZuI
AZuI
ACGTACTAAGCTCTCATGTTTAACGAACTAAACCCTCATGGCTAACGTACTAAGCTCTCATGGCTAACGTACTAAGCTCTCATGTTTCACGTACTAAGCTCTCAT TGCATGATTCGAGAGTACAAATTGCTTGATTTGGGAGTACCGATTGCATGATTCGAGAGTACCGATTGCATGATTCGAGAGTACAAAGTGCATGATTCGAGAGTA 200
180
160
140
120
100
AZI
GTTTGAAC* CAAACTTG
AAA
hfr~~~~~~~tLL ~~~~~~HindIII
TAGCCTCTA AGGTTTTAAGTTTTATAAGAAAAAAAAGAATATATAAGGCTTTTAAAGCTTTTAAGGT AiATAT*ATCAGCAACTT4 TTATATJTAGTCGTTGAAT TATCGGAGAT CAAAATTCAAAATATTCTTTTTTTTCTTATATATTCCGAAAATTTCGAAAATTCCA
TUATTTA80
60
40
20
1
EaoRII HaeII MZI TTAACGGTTGTGGACAACAAGCCAGGGATGTAACGCACTGAGAAGCCCTTAGAGCT-TCAAAGCAATTTTCAGTGACACAGGAACACTTAACGGCTGACA .... 3' ATACAT TGCTGACTCTTCGGGAATCTCGGAGAGTTTCGTTAAAAGTCACTGTGTCCTTGTGAATTGCCGACTGT.... 5 -20 -40 -60 -80 -100 FIG. 5. Nucleotide sequence of the 520-bp segment of plasmid pRK526. Bases are numbered from the HindIII site, and restriction enzyme sites are indicated. Arrows show the 22-bp direct repeats. Regions of 2-fold rotational symmetry are indicated by arrows between strands. The bracketed bases indicate a possible RNA polymerase binding site (promotor); the positions of the tentatively identified methylated cytosine residues are indicated by asterisks.
are interrupted by nonsense codons in both directions, indicating that no large polypeptides are coded for in this region. However, coding for small functional peptides cannot be ruled out. An exception is a possible GUG start at base 370, which is open with respect to translation as far as the sequence has been determined. Seven tandem 22-bp direct repeats are designated by arrows in Fig. 5 starting at position 93 and continuing through 246. An eighth repeat unit in the region 362 to 383 contains the open GUG start. As shown in Fig. 6, the repeat units are nearly-identical in sequence, with the exception of the middle unit which contains five different bases. DISCUSSION The nucleotide sequence of the region containing a functional origin of plasmid R6K replication that is present in pRK526, a low molecular weight derivative of plasmid R6K, has been determined. The replication of pRK526 in E. coli requires the presence of HindIl fragments 9 and 15 of R6K, which code for a protein (Xr) that is required for the initiation of R6K replication (6-8). These fragments can be incorporated into E. coli cells by joining them to a ColEl replicon or to a X phage derivative that is integrated in the host chromosome (8). Plasmid pRK526 is maintained in cells carrying HindIII fragments 9 and 15 at a copy number similar to that of the parent R6K plasmid (10-15 copies per chromosome) (8). The functional origin of replication in pRK526 described in this study is contained within portions of HindIll fragment 9 and the adjacent HindIll fragment 4 (8). The close proximity of a gene speci5'
AACGTACTACDC®CTCATGTTT
3'
TI T (DA C G T A C T A A S C T C T C A TIG I I
QA C G T A C T A A G C T C T C A G T T AACGTACTAAGCTCTCATGTTT A A C GSA C T A A®C®C T C AT GGT A A C G T A C T A A G CT C T C A GZI T AACGTACTAAGCTCTCATGTTT (A C GST A C T A A G C T C T C A TIG T I T
FIG. 6. Nucleotide sequence of the eight 22-bp direct repeats which are read from left to right in Fig. 5. The top repeat unit is separated from the seven tandem repeats and spans bases 362-383. Circled residues indicate differences between bases.
fying a protein required for the initiation of DNA replication to the origin of replication has been shown for several different replicons. Examples of this close spatial relationship are the cisA protein of phage qX174 (20-22), the 0 protein of phage X (23-25), and the T antigen of the animal virus simian virus 40 (26-29). In the case of the R6K derivatives described in this study, evidence has been obtained for the start of translation of the ir protein in HindIII fragment 9 (unpublished observations). The nucleotide sequence obtained for a portion of fragment 9 contains a possible promoter for leftward transcription of the pir gene (Fig. 5). This site contains an RNA polymerase recognition sequence (nucleotide bases 49 through 60) within a region having limited 2-fold rotational symmetry. The sequence -T-A-T-A-T-T-A- at positions 74 through 80 differs by one base from the one described by Pribnow as an RNA polymerase binding site (18). If this is the case, transcription would begin at position 86, proceed through the region containing the direct repeats, and continue through the rest of fragment 9 and all of fragment 15 to include the extra pir structural gene. Binding of the or protein to the direct repeat region (a presumptive operator) could serve to regulate gx synthesis by shutting off transcription. This would provide an autoregulatory mechanism for the regulation of DNA replication and possibly plasmid copy number. The finding of a similar copy number of plasmid derivatives carrying this origin fragment in E. coli cells carrying the pir gene either on the chromosome or on a multicopy plasmid is consistent with an autoregulation of ir protein synthesis (8). Direct repeats function in regulating the expression of the cI and crc genes in phage A (30, 31). The CI and Cro repressor proteins bind to the 17-bp repeats that constitute the left- and right-hand operators and promoters of the phage (32). It has been proposed that this binding serves to autoregulate transcription of these repressor genes (32). Tandem repeats also have recently been observed at or near the origins of DNA replication of phage A (33) and simian virus 40 (29). It is also possible that the direct repeats found near the functional origin of plasmid R6K are involved in regulating other properties of this plasmid such as incompatibility or segregation or both. Initiation of R6K DNA replication is rifampicin sensitive, indicating an involvement of RNA polymerase (6, 7). The fact
1154
Biochemistry: Stalker et al.
that pRK526 is not maintained in E. coli cells if a DNA fragment is inserted in the HindIII site at the 9/4 junction raises the possibility that an origin RNA primer is synthesized across this HindIII site. RNA polymerase conceivably could initiate this RNA primer synthesis in the vicinity of the EcoRII site at nucleotide positions -20 to -34, which contain an RNA polymerase recognition sequence (Fig. 5). The primer would then be synthesized across the 9/4 junction and possibly terminate at the stretch of adenine residues at positions 21-29 (Fig. 5). Adenine residues have been implicated in rho-independent termination of transcription by RNA polymerase (19). Runs of adenine residues of varying lengths (five to nine bases) also have been observed near origins of replication of plasmid ColEl (34, 35), phage X (33), the papovaviruses simian virus 40 (28. 29), polyoma (36), and BK (37), the origin of E. coli replication (38, 39), and the leader sequence of the tryptophan operon (40). The significance of the putative methylated cytosine residues at positions -33 and -35 with regard to the replication origin activity of this region is not known. These modified bases are part of an EcoRII restriction endonuclease cleavage site (41) and are recognized by both the EcoRII and cellular methylase activities (42). Methylated cytosine residues are found in the region of the origin for replication of plasmid CoWEl, but in this case methylation of these bases is not required for maintenance of the plasmid. It is unclear whether the origin fragment of R6K whose sequence we have determined corresponds to either the a or A origins previously mapped by electron microscopy (3, 5). Attempts to isolate other origin-containing fragments from R6K by the transcomplementation system have proven unsuccessful. It is clear, however, that the nucleotide sequence described contains an origin of replication in the region of the HindIII site that is functional in the presence of ir protein. Whether the ir protein recognizes a sequence of nucleotides at this origin or interacts with another polypeptide involved in the initiation event at this sequence remains to be determined. We are deeply indebted to T. Friedman, P. Laporte, and P. Deinenger for introducing us to sequencing techniques and providing facilities for the initial experiments and helpful suggestions. We are grateful to M. Inuzuka and M. Kahn for helpful discussions and critical reading of the manuscript. This work was supported by grants from the National Institute of Allergy and Infectious Diseases (AI-07194) and National Science Foundation (PCM77-06533). D.S. was supported by a U.S. Public Health Service Postdoctoral Fellowship (1-F32GM06542). R.K. is the recipient of a University of California Predoc-
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