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Origins of Replication of the Plasmids Cole1 and Pbr322 (Site Specificity/ATP Hydrolysis/Initiation of DNA Synthesis) S

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Origins of Replication of the Plasmids Cole1 and Pbr322 (Site Specificity/ATP Hydrolysis/Initiation of DNA Synthesis) S Proc. Natl. Acad. Sci. USA Vol. 77, No. 11, pp. 6521-6525, November 1980 Biochemistry Identification of two Escherichia coli factor Y effector sites near the origins of replication of the plasmids ColE1 and pBR322 (site specificity/ATP hydrolysis/initiation of DNA synthesis) S. L. ZIPURSKY AND K. J. MARIANS Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461 Communicated by Jerard Hurwitz, August 11, 1980 ABSTRACT The Escherichia coli replication factor Y has MATERIALS AND METHODS been characterized as a OX174 (+) strand specific DNA-de- pendent phosphohydrolase. In conjunction with other E. coil Enzymes and Assay Conditions. Factor Y was purified replication proteins, factor Y is involved in the formation of according to the procedure of Wickner and Hurwitz (10), using heterogeneous primers that are elongated by the E. coli DNA as an assay the differential ATPase activity of factor Y with polymerase III elongation machinery. We report here that the cX174 and bacteriophage fd DNAs as effectors. Fraction VII heat-denatured DNAs of plasmids pBR322 and CoIEl serve as (glycerol gradient) was used for all experiments described in effectors for the hydrolysis of ATP by factor Y. The DNA se- this report. One unit of ATPase activity corresponds to the quences of pBB322 responsible for factor Y effector activity have cleavage of 1 ,umol of ATP to ADP and in 30 min at 30'C been localized. Two separate regions of the pBR322 chromo- Pi some support Y ATPase activity. These sequences are near the with OX174 DNA as effector. The specific activity of fraction replication origin and are located on opposite DNA strands. VII was 320 units/mg and represented a 2000-fold purification from the ammonium sulfate fraction. Assay for factor Y ATPase In Escherichia colh three enzymatic mechanisms have been activity was as described (10), with the addition of E. coli characterized that could account for de novo priming on the ssDNA-binding protein (DBP) (a gift of Jack Chase of this in- lagging strand. A primer could be formed by RNA polymerase stitution) at a ratio of 1 ,ug of DBP per 180 pmol of nucleotide (1-3), by the dnaG gene product acting alone at a unique residues (11). The concentration of DNA in assays was main- chromosomal site (4-6), or by the dnaG gene product directed tained at 0.36 nM with respect to molecules. Standard assays to the lagging strand template by factor Y (7, 8). were 25 ,ul and were incubated at 30'C for 30 or 60 min. The In dtro DNA replication of recombinant DNA plasmids specific activity of the [y-32P]ATP used varied from 10 to 50 containing the leading strand origin of bacteriophage bX174 cpm/pmol. Pi was determined by the method of Conway and RFI (replicative form I) DNA suggested the presence of factor Lipmann (13). Y DNA Restriction enzymes were purchased from New England effector sites on plasmid pBR322 (9). Biolabs and were assayed in the buffers suggested by the Factor Y described by Wickner and Hurwitz (10) and by manufacturer. Bacterial alkaline phosphatase (BAPF) was from Shlomai and Kornberg (11) (n' in their nomenclature) is a Worthington and bacteriophage T4-polynucleotide kinase was OX174 specific DNA-dependent ATPase. The latter group from P-L Biochemicals. identified a 54-nucleotide region of the OX174 viral strand that Preparations of DNAs. kX174 and fd (+) strand DNAs were functioned as an effector for factor Y ATPase activity. Pre- purified as described (14). kX174 RFI [3H]DNA was prepared sumably factor Y directs the priming proteins involved in from OX174 am3-infected E. coli HF4704 in the presence of kX174 single-stranded DNA (ssDNA) RF replication to a chloramphenicol at 30 ,ug/ml (9). pBR322 and ColEl unique chromosomal site (11) from which a "mobile primase" [3H]DNAs were prepared from the E. coil strains CR34 and apparatus is assembled that is capable of synthesizing hetero- JC411, respectively, after amplification overnight in the pres- geneous primers complementary to contiguous DNA sequences. ence of chloramphenicol at 180,ug/ml (9). Bacteriophage fi (12). RFI DNA was the gift of P. Model of the Rockefeller University. We report here that DNAs of both pBR322 and the related DNA fragments were purified preparatively from polyacryl- plasmid CoIEl contain two discrete chromosomal segments that amide gels by electroelution followed by dialysis overnight when denatured support factor Y ATPase activity. Our analysis against 10 mM Tris-HCI, pH 8 at 4°C/10 mM NaCl/1 mM indicates that these segments are located near the plasmid origin EDTA (TEN buffer). Residual acrylamide was removed by of DNA replication. Both segments are also downstream from centrifugation in an Eppendorf microcentrifuge. the replication origin; one is located on the L strand and the other further downstream on the H strand.* We suggest that Abbreviations: Factor Y, E. coli replication factor Y (10); RF, circular the site located on the L strand functions as an origin for lag- duplex DNA molecule (replicative form); RFH, negatively supercoiled ging-strand DNA synthesis in pBR322 replication and that the circular duplex DNA molecules; ssDNA, single-stranded DNA mole- site located on the H strand is involved in converting leading- cule; DBP, E. coil ssDNA-binding protein; (+) strand, viral strand; (-) strand DNA synthesis from a continuous to a discontinuous strand, complementary strand; R-loop, structure in which a "bubble" is formed in duplex DNA by hybridization of an RNA segment to one mode. strand of the duplex. * We refer to the pBR322 DNA leading and lagging strand templates The publication costs of this article were defrayed in part by page as H and L, respectively. In ColEl replication the leading strand charge payment. This article must therefore be hereby marked "ad- template is the H strand and the lagging strand the L strand. The H vertisement" in accordance with 18 U. S. C. §1734 solely to indicate and L designations of the two strands of ColE1 reflect heavy and light this fact. density, respectively. 6521 Downloaded by guest on September 30, 2021 6522 Biochemistry: Zipursky and Marians Proc. Natl. Acad. Sci. USA 77 (1980) RESULTS Table 1. Factor Y ATPase activity with denatured DNA Factor Y ATPase Activity Dependent on Denatured 4X174 fragments RFI and pBR322 DNAs. As shown in Fig. 1, the ATPase ac- 32pi formed, tivity of factor Y using effector DNA saturated with DBP was DNA effector added nmol highly specific for q5X174 (+) strand DNA compared to fd (+) strand DNA. An identical specificity was evident when heat- OX174 RFI 9.72 denatured RF1 DNA was compared to denatured fI RFI fl RFI 0.10 OX174 pBR322 5.95 DNA. Equimolar amounts of 4X174 (+) strand DNA and de- HindII + Pvu II fragments of pBR322 natured OX174 RFI DNA gave identical rates of ATP hydrolysis A (2065-3906) 3.03 by factor Y (data not shown). Denatured pBR322 DNA sup- B (650-2065) 0.12 ported significant ATPase activity in the presence of factor Y. C (3906-650) 0.12 The major difference between the activity observed with Pst I + Pvu II fragments of pBR322 OX174 RFI and pBR322 DNAs was that the shape of the curve A (3608-2065) 0.09 with the latter DNA was sigmoidal. The reason for this is not B (2065-3608) 2.77 clear, but the shape may reflect differences in the affinity of Hae II fragments of pBR322 factor Y for different effector sites. GolEl DNA gave results A (2718-232) 0.07 essentially identical to those with pBR322 DNA (data not B (1726-2348) 4.29 shown). The ATPase activities with denatured 4X174 RFH DNA E (2348-2718 2.87 and pBR322 DNA were identically sensitive to N-ethylmalei- Hae II fragments of ColEl mide and heat (data not shown). A 0.08 Two Regions of pBR322 and ColEl DNA Support Factor B 0.15 Y ATPase Activity. To identify the region of the pBR322 and C 2.14 ColEl genomes that supported factor Y ATPase activity, we D 0.10 purified a series of DNA fragments of plasmids generated with E 4.53 restriction endonucleases. Denatured fragments were assayed F ND for their ability to support factor Y ATPase activity. The results The indicated DNA preparations and DNA fragments were heat of these experiments are shown in Table 1. denatured. Reaction mixtures (25 ,ul) contained 18 fmol of single- Digestion of pBR322 DNA with a combination of the HindIl stranded molecules with 0.08 unit of factor Y and were incubated for and Pvu II endonucleases yields three fragments, only one of 60 min. DNA fragments were prepared as follows: various amounts (10-30 ug) of pBR322 [3H]DNA (5.7 cpm/pmol) and ColEl [3H]DNA which, the A fragment, had effector activity when denatured. (0.57 cpm/pmol) were digested overnight with the indicated restriction enzyme or combinations of enzymes. The digests were heated at 650C for 10 min, extracted with phenol, and precipitated with ethanol. The regions of the gel containing the fragments were excised and the DNA was recovered by electroelution. DNA concentrations were calculated A from the specific activity of the starting DNA. The numbers in pa- rentheses after each pBR322 DNA fragment are the positions of the 15 5' ends of the recognition sequence that bracket the fragment. See ref. 15 for the Hae II cleavage map of CoIEl DNA. ND, not determined. A Similarly, only one of the two fragments (fragment B) from a double digestion of pBR322 DNA with the endonculeases Pst 0~~ I and Pvu II supported factor Y ATPase activity when dena- 10 tured.
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