Jun 16, 1980 - The recombinant superhelical templates are similar to OXDNA but radically different from pBR322 DNA in that: (i) theyare cleaved by the OX A ...
Proc. Nati. Acad. Sci. USA Vol. 77, No. 9, pp. 5182-5186, September 1980
Biochemistry
In vitro DNA replication- of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage 4X174 (+X174 A protein/circular OX174 DNA/leading and lagging DNA strand synthesis)
S. L. ZIPURSKY, D. REINBERG, AND J. HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461
Contributed by Jerard Hurwitz, June 16, 1980
ABSTRACr The origin of phage OX174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for #X174 RHi DNA as template in XX174-specific reactions in vito. We have shown that the recombinant plasmids: (i) are cleaved by the *X174 A protein; (ii) support net synthesis of unit-length single-stranced circular DNA in the presence of the $X174 A protein and Escherichia cofl rep protein, DNA-binding protein, and DNA polymerase m elongation system; (iii) support replication of duplexes catalyzed by the X174 A protein ana crude extracts of E. coli. The phage 4X174 (OX) A protein initiates RF - RF replication on superhelical OX replicative form I (RFI) by introducing a site-specific endonucleolytic cut in the viral strand (1-4). The phage viral strand (+ strand) is recognized as substrate for endonucleolytic cleavage when in the single-strand configuration or the negatively supercoiled form but not when in the relaxed duplex form (1, 5). The OX A protein cleavage results in a free 3'-OH terminus and an apparent covalent attachment of the protein to the newly generated 5'-phosphate end (6-8). In conjunction with Escherichia coli host factors, the nicked duplex DNA-protein complex can be utilized as template for the production of double-stranded RF progeny and the synthesis of (+) single-stranded circular DNA [ss(c)] (3,4, 9). The OX A protein is implicated in the terminal ligation step and in aiding the E. coli rep protein in unwinding the double helix in advance of the replication fork (10). The apparent multifunctionalism of the OX A protein is dependent upon a short segment of the OX chromosome-the origin of viral strand synthesis. Recombinant DNA technology and in vitro mutagenesis have contributed to the assignment of essential sequences within replication origins (11-13). These experiments, however, depend upon the feasibility of altering the origin sequence and assessing its replicative capacity in vivo. Two features of OX progeny RF synthesis severely limit the potential of this approach. First, the origin sequence is located within the OX A cistron and second, the OX A protein is cis acting in vlvo (14, 15) [but not in vitro (8)]. The availability of in vitro replication systems specific for OX RFI provide a functional assay for the structural requirements of the OX RF replication origin. In order to perform these experiments, it was necessary to introduce the OX duplex replication origin into a heterologous background and clone it. We report here the cloning of the 4X progeny RF replication origin into the plasmid pBR322 and its template activity in in vitro replication systems specific for AX RFI DNA.
MATERIALS AND METHODS Bacteria and Viruses. E. coli strains and their relevant genotypes were: H514 (OX174s, endA), MV12 [(PLC 44-7)/ colEl(PLC 44-7) rep+], HMS 83 (polA, polB), HF4704 (OX174s), D92 (OX174S, rep-), PC22 (polA, dnaC), BT1029 (endA, polA, dnaB), SK2267 (F-, thi, gal, endA, sbcB, recA, hsdR4, tonA), CR34 (thr, leu, thi, supE, lac, tonA, thyA, dra, rpsL). Phage used in all experiments was 4X174 am3 (lysis-, gene E). Preparation of Enzymes, Protein Fractions, and DNAs. Ammonium sulfate fractions from E. coil (referred to as receptors) were prepared by a modified version of a published procedure (3). Crude extracts passed through DEAE-cellulose [previously referred to as fraction II (3)] were prepared from infected and uninfected cells as described by Yasumoto et al. (3). The OX A protein (8), E. coli rep protein (16), E. coil DNA-binding protein (DBP) (17), DNA elongation factor I (unpublished procedure), and DNA polymerase III (unpublished procedure) were prepared as described (or with modifications) from kX174 am3-infected E. coil H514, E. coli MV12/ColEl(PLC 44-7), E. coil CR603/(pDR 2000), E. coli HMS 83, and E. coil HMS 83, respectively. In experiments described below, phosphocellulose fractions of the OX A protein and the DNA-cellulose fraction of the rep protein were routinely used. The E. coli DBP used in these experiments was a gift from J. Chase. OX RFI [3H]DNA was prepared from 4X174 am3-infected E. coil HF4704 in the presence of chloramphenicol at 30 /g/ml (1). Plasmid pBR322 was prepared from E. coil SK2267 transformed cells as described (18). Plasmids used for DNA templates in replication assays (PBR322 and recombinants G5 and G39) were labeled with [3H]thymidine and were isolated from either transformed E. coli strains SK2267 or CR34. These DNAs were treated with pancreatic RNase A (2 Ag/A26o unit) for 60 min at 30'C before CsCl/ethidium bromide density gradient centrifugation. In addition, these DNAs were purified through high-salt (1 M NaCl) 5-20% sucrose gradients. Techniques Used in Cloning DNA. E. colt strains SK2267 or CR34 were transformed as described (19). Restriction enzymes were purchased from Bethesda Research Laboratories (Rockville, MD) or New England BioLabs and phage T4 DNA ligase was purchased from New England BioLabs. T4 polynucleotide kinase was a gift from Heike Pelka of this departAbbreviations: OiX, phage OX174; DBP, E. coli DNA-binding protein; Tets, tetracycline sensitivity; Ampr, ampicillin resistance; RFI, negatively supercoiled covalently closed double-stranded circular DNA molecules; RFII, double-stranded circular DNA molecules containing a discontinuity in one strand; ss(c), single-stranded circular monomer-length DNA; ss(l), single-stranded linear monomer-length
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DNA.
this fact.
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Biochemistry: Zipursky et al. ment. The BamHI linker, 5'-HO-C-C-G-G-A-T-C-C-G-GOH-3', was a gift from K. Marians of this department'. XX A Protein Endonuclease Assay. Endonucleolytic activity of the OX A protein was measured as follows: Reaction mixtures (50,l) containing 0.058 pmol (as molecules) of RFI (plasmids or OX DNA), 20mM Tris-HCI at pH 7.5,4mM dithiothreitol, 10 mM MgC12, heat-denatured bovine serum albumin at 0.1 mg/ml, and indicated levels of OX A protein were incubated at 30'C. Reactions were terminated with 10mM EDTA, 0.1% sodium dodecyl sulfate, and proteinase K at 0.1 mg/mil followed by incubation at 370C for 1-2 hr. The DNA was then subjected to electrophoresis in1% agarose gels (4 mm thick, 10cm long) run at 10 V/cm for 90 min in TEA buffer (50mM Tris/40mM sodium acetate/imM EDTA, pH 7.9). Gels were stained with ethidium bromide (1 Mg/ml) for 10 min and photographed as described (20). For further quantitation, DNA was eluted from agarose gels by heating at 100C in1 ml of distilled water and radioactivity was measured in Aquasol. Synthesis of ss(c)AX DNA from RFI DNA. Reaction mixtures (50,u) containing 20mM Tris-HCl at pH 7.5, 10mM MgCI2, 4mM dithiothreitol, 40 AM [3H]dTTP or [a-32P]dTTP (400-1500 cpm/pmol), 40 ,M each of dATP, dCTP, and dGTP, rifampicin at 10 Mg/ml, 2mM ATP, 0.036 pmol of RFI DNA molecules, 1 unit of OX A protein, 0.026 unit of E. col rep protein, 6.7 units of DNA polymerase III (containing dnaZ and DNA elongation factor III), 0.3 unit of DNA elongation factor I, and 0.4 Mg of E. colh DBP were incubated as indicated. Reactions were terminated by acidification and acid-insoluble radioactivity was measured. Products formed in the above reactions were analyzed by stopping the reactions with 1 mM EDTA, 0.1% sodium dodecyl sulfate, and proteinase K at 0.1 mg/ml followed by incubation at 370C for 90 min. The mixture was extracted twice with chloroform/phenol (1:1, vol/vol) and precipitated with 3 vol of ethanol. The products were subjected to electrophoresis on 1.5% agarose gels (4 mm thick, 10 cm in length) in TBE buffer (0.1 M Tris/0.1 M boric acid/i mM EDTA, pH 8.5) at 10 V/cm. The gels were stained with ethidium bromide and photographed, and RF values were determined for markers (20). RF values for labeled products were determined by radioautography. Markers for recombinant single-stranded linear [ss(l)] and ss(c) molecules were derived from heat-denatured recombinant RFII molecules prepared as described (21). OX RF - RF Synthesis. Reaction mixtures (50 Ml) containing 20 mM Tris-HCl at pH 7.5, 10 mM MgCl2, 4 mM dithiothreitol, 40 MM [3H]- or [a-32P]dTTP (350-4000 cpm/ pmol), 40MuM each of dATP, dGTP, and dCTP, 2mM ATP, 0.1 mM each of UTP, GTP, and CTP, rifampicin at 10,Mg/ml, 0.1 mM NAD+, 0.013-0.04 unit of dnaC protein, 0.01-0.02 unit of dnaB protein, 0.17 unit of OX A protein, ammonium sulfate fraction (120,Mg/protein), and 0.125-0.75 ,g of RFI DNA (OX or recombinant DNAs) were incubated for 30 min at 30C and radioactive acid-insoluble material was measured. Further product analysis was carried out as described for ss(c) synthesis except that the proteinase K treatment was omitted. RESULTS RF Replication Origin. The OX Progeny Cloning of the OX progeny RF replication origin is located within the OX Hae III fragment 6b (6). Double digestion of OX RFI DNA with both HinfI and Hae III restriction enzymes facilitated isolation of fragment Hae III-6b free of fragment Hae III-6a. The synthetic self-complementary decanucleotide 5'-C-C-G-G-A-T-C-CG--3' (which in duplex form contains a BamH restriction site) was 5'-end-labeled with T4 polynucleotide kinase, annealed, and blunt-end ligated to Hae III-6b with T4 DNA ligase (Fig.
Natl. Acad. Sci. USA 77(1980) 5183 1). The fragment containing the newly added BamiHI restriction sites at each end was treated with BamHI endonuclease Proc.
to generate overlapping complementary ends. The plasmid pBR322 DNA, linearized with BamHI endonuclease and
treated with bacterial alkaline phosphatase, was linked with T4 DNA ligase to the BamHI sites on the Hae III fragment 6b. The final ligated product was used to transform E. coliSK2267; transformants with tetracycline sensitivity and ampicillin resistance (TetV Ampr) phenotype were selected. Analysis of plasmid DNA from selected transformants yielded bacterial clones in which the OX DNA Hae III-6b fragment was integrated into the plasmid in two different orientations (Figs. 1 and 2). These recombinant plasmids are referred to as G5 and G39. Endonucleolytic Cleavage of Recombinant Plasmid RFI DNA by the OX A Protein. The OX A protein site-specific endonuclease activity is a necessary prelude to other reactions in which the OX A protein is required. Consequently, we analyzed the interaction of the OX A protein with the two recombinant DNAs, G5 and G39. The rate (data not shown) and extent of cleavage of the recombinant DNA molecules were the same as those found with kX174 am3 RFI DNA (Fig. 3). In no case was OX A protein-mediated cleavage of pBR322 DNA observed. ss(c) Synthesis with Purified Proteins. Eisenberg et al. (10) demonstrated that the OX A protein in conjunction with E. coli rep protein, DBP, and the DNA polymerase III elongation
system catalyzed OX RFI-dependent synthesis of (+) strand ss(c). In addition to being required for initiation, the OX A protein may also be involved in elongation and circularization of the displaced strand. We reasoned that these replicative functions would depend upon a relatively short nucleotide sequence; synthesis initiated at the OX A cleavage site on a circular recombinant molecule would proceed through contiguous plasmid sequences and terminate within the cloned OX phage DNA sequence. The recombinant DNAs but not pBR322 DNA substituted for OX RFI DNA in the in vitro (+) strand synthesizing system. Synthesis supported by recombinant DNAs: (i) pBR322
Hoae
*X174 6b +
Bom
Linker
~~~~~BornHI
R
Mix in Presence of T4 ligose ffR
t
ChampR
Barn HI
Recornmon/t Molecule
Recombinant 5' H 3' L 65
/ Bom H tetR BaM H I \ Iu I Alu/I i/174 repl\ati\ 3' 5 Al1
Alu I AluI
Recombinant 5'H /
G39
I
-- -
#X174 repicotion (+)-
3'L
t
Alu
I3' \ 5
Alul
XX
FIG. 1. Cloning of the viral strand origin. OX fragment Hae III-6b was purified and modified as described in the text. Step 1 in-
volved transformation, selection of Ampr transformants, screening for Tets transformants, and isolation of plasmids from Ampr Tet8 cells. Isolation of plasmid DNAs demonstrated the two different orientations 2 and 3. The L strand is the leading strand of pBR322 replication (22) and the H strand is its complementary strand. The symbol (+) is defined as the OX174 viral strand. The arrows indicate the direction of XX A protein-dependent DNA synthesis.
5184
Biochemistry: Zipursky et al.
Proc. Nati. Acad. Sci. USA 77 (1980)
_60
-
25040
-
U30
-
0
a 20
~i0 0.5 1.0 1.5 2.0 ,X A protein added, units FIG. 3. Cleavage of RFI DNAs by XX A protein. DNA preparations were incubated with various amounts of the OX A protein. Cleavage of G39 RFI DNA was monitored by agarose gel electrophoresis followed by ethidium bromide staining. This DNA and G5 RFI DNA were cleaved at identical rates. A, OX RFI [3HJDNA; 0, recombinant plasmid G5 RFI [3HJDNA; *, vector pBR322 RFI
[3H]DNA.
FIG. 2. Orientation of OX progeny RF replication origin in recombinant DNAs. Lanes 1, 2, and 3 are Alu I digests of 1 Ag of G5, pBR322, and G39 RFI DNAs, respectively. Inserts are located in pBR322 DNA Alu I fragment 3 (655 base pairs). The OX Hae III fragment 6b contains one Alu I cleavage site. Loss of pBR322 Alu I fragment 3 in the recombinants results in the appearance of two new fragments whose lengths depend upon the orientation of the inserted Hae III fragment 6b. Alu I cleavage of G5 results in new fragments of 501 and 441 base pairs. Similar cleavage of G39 results in new fragments of 534 and 409 base pairs. Digests were run on a 5% acrylamide gel (30:1 acrylamide to bisacrylamide) for 50 min at 35 W in 0.05 M. Tris/0.05 M boric acid/0.5 mM EDTA, pH 8.5.
required the same enzymes necessary for OX DNA synthesis (data not shown), (0) produced recombinant ss(c) (Fig. 4), and (ill) occurred at 70% of the rate observed with equimolar amounts of OX RFI DNA (Fig. 5). Because the OX origin sequence is inserted into pBR322 DNA in two orientations, the ss(c) products of G5 and G39 RFIdependent DNA synthesis are different. One can infer from the orientation data that in the recombinant plasmid G5 the OX (+) strand is covalently joined to the L-strand of pBR322 DNA, whereas in G39 the OX (+) strand is ligated to the pBR322 H strand. The products, therefore, of (+) strand DNA synthesis supported by the G5 and G39 RFI templates are OX (+), pBR322 (L) and OX (+) pBR322 (H), respectively. The sequences of the recombinant products were verified by hybridization to the separated strands of pBR322 HMnfl fragment 6 (data not shown). Replication of RF Products. Replication of the RFI recombinant DNAs was examined with receptor extracts of E. capable of synthesizing OX RF DNA. Synthesis of OX RF DNA requires the OX A protein, rep protein, the DNA polymerase III elongation system, the products of the E. coil genes
coil
dnaB, dnaC, and dnaG, and a number of other E. coli-encoded proteins (23). This synthesis is sensitive to RNase A and to the DNA gyrase inhibitors coumermycin and nalidixic acid (23). Data in Table 1 summarize the template activity of the recombinant DNA preparations containing the OX viral strand origin. Insertion of the restriction fragment Hae III-6b into pBR322 DNA permitted rifampicin-resistant replication of the plasmid by the cell-free system. In contrast, pBR322 RFI DNA was inactive as template over a wide concentration range. As in the case of OX RFI, replication of the recombinant DNAs required the OX A protein and was inhibited by coumermycin, nalidixic acid, and RNase (23). Although the replications of XX RFI and recombinant RFI DNAs were similar, significant differences were observed. Products formed with G5 and G39 DNA templates were predominantly RFI and RFII, whereas products synthesized from OX RFI DNA were a mixture of RFII, RFI, and ss(c) (Fig. 6). DNA synthesis with OX RFI and plasmid RFI DNAs was sig-
V:~~~~~~~~~~~~~~~~~~~~11
FIG. 4. Agarose gel electrophoretic analysis of products formed in OX174 and recombinant ss(c) synthesis. Lane a, OX product; lane
b, G5 recombinant product; lane c, G39 recombinant product.
Biochemistry: Zipursky et al.
Proc. Natl. Acad. Sci. USA 77 (1980) "I
N_ IFII
RFKI
20
(I
c
I
RFX1I-
0
h)
(
r
I
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i
( ;:s9
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; 10
20
30 40 Time, min
90
FIG. 5. Rate of ss(c) DNA production. The synthesis of ss(c) DNA was carried out as described in Materials and Methods except that reaction volumes were doubled and 10-A1 aliquots were removed at the times indicated to follow dNMP incorporation. DNA was synthesized with kX RFI (@), G5 RFI (0), and G39 RFI (-).
moidal with respect to DNA concentration (Fig. 7); the degree of sigmoidicity was more pronounced with both recombinant DNAs than with qX RFI. We have shown that the difference was not due to inhibitors present in the plasmid DNAs (data not shown). DISCUSSION We have demonstrated that recombinant superhelical plasmids that contain the replication origin of 4X174 progeny RF synthesis and the entire sequence of pBR322 DNA support DNA synthesis in vitro in replication reactions specific for OX RFI. The recombinant superhelical templates are similar to OX DNA but radically different from pBR322 DNA in that: (i) they are cleaved by the OX A protein; (Ui) they support net synthesis of ss(c) DNA molecules catalyzed by the OX A protein, rep protein, DBP, and the DNA polymerase m elongation system; (ii) they support synthesis of RFi and RFM in cell-free extracts that replicate OX RFI. It has been shown (24-26) that the OX A protein cleaves RFI molecules of OX-related icosahedral phages G4, ST-1, aS, and OK. Sequence analysis of G4 and ST-1 DNAs (24,25) established Table 1. Requirements for RF replication dTMP incorporation with RFI, pmol Reaction mixture X G-39 G5 260 130 118 Complete 2 8 3 -,XX A protein - dnaB protein 14 87 20 -dnaC protein 24 171 28 - dnaB and dnaC proteins 7 78 10 + RNase A (1 ,g/ml)2 4 18 + novobiocin (300 jsg/ml) 12 55 9 + nalidixic acid (300,ug/ml) 12 9 35 Omit DNA
