Proc. Natl. Acad. Sci. USA Vol. 73, No. 6, pp. 1887-1891, June 1976
Biochemistry
DNA synthesis in vitro dependent upon kX174 replicative form I DNA (OX A gene product/DNA replication) CHIKAKO SUMIDA-YASUMOTO, ARTURO YUDELEVICH, AND JERARD HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461
Contributed by Jerard Hurwitz, March 17,1976
Cells were grown and infected with phage at 300 for E. coli dna temperature-sensitive (ts) mutants or 350 for other E. coli strains, unless noted. Cells were grown to an OD65o of 0.3 in medium supplemented with 4 jig/ml of thymine (19), infected with OX wild type or mutants with 5-10 phages/bacterium, and incubated for 5 min without shaking to allow adsorption. CaCd2 was then added (final concentration 1 mM) and infection continued as indicated in figure and legends. Cells were collected and frozen (3) and stored at -80° for at least 1 month without loss of XX RF synthetic activity. Preparation of fraction II is detailed in the legend of Table 1. Assay of DNA Synthesis. Standard reaction mixtures (0.05 ml) contained, in order of addition, 20 mM Tris.HCl (pH 7.5), 10 mM MgCl2, 4 mM dithiothreitol, 1 mM ATP, 1 mM NAD+, 40 AM each of dATP, dCTP, dGTP, and [a-32P]dTTP (200-700 cpm/pmol), 100MuM each of CTP, UTP, and GTP, 1.8 nmol of OX [3H]RFI, (6 cpm/pmol), 5 Al of ammonium sulfate fraction from uninfected cells (protein as indicated), and fraction II (prepared from OX-infected cells described below). Reaction mixtures were incubated for 40 min at 300 (unless specified), and acid-insoluble radioactivity was determined (3). When products were characterized by sedimentation analyses, samples were treated either as described by Sakakibara and Tomizawa (20) or by addition of EDTA (24 mM) and Sarkosyl (final 2%) followed by incubation at 420 for 10 min.
Extracts of Escherichia coli strains infected ABSTRACT with bacteriophage 4X174 catalyze DNA synthesis dependent on double-stranded, circular kX174 replicative form I (OX RFI) by a semiconservative process. The reaction required Mg++, ATP, all four dNTP, and exogenous XX RFI DNA as teaplate and yielded OX RFI and OX RFII. The reaction was inhibited by nalidixic acid and novobiocin but not by rifampicin. DNA synthesis required the OX174 gene A product and E. coli gene products dnaB, dnaC(D), dnaG, and rep.
Replication of /X174 single-stranded, circular DNA occurs in three stages: synthesis of (a) parental replicative form (RF), (b) progeny RF, and (c) progeny single-stranded, circular DNA. In stage a, parental RF synthesis depends solely upon host proteins, and a cell-free system catalyzing this reaction has been developed (1, 2). Complementation assays with this system allowed the isolation of Escherichia coli dnaB, C(D), G, and Z gene products (3, 4) and other proteins required for conversion of OX174 DNA to RFII (5-7). This system is specific for OX174 DNA and is inactive with duplex OXRF forms. In avo, progeny RF and/or progeny qX174 DNA synthesis also depends upon E. coli dnaB (8), dnaC(D) (9), dnaE (9), dnaG (10), dnaZ (11, 12), and rep gene (13) products in addition to the kX174 gene A product (14). To study the mechanism of double-stranded DNA replication, we have developed a cell-free system dependent upon added OX RFI DNtA. This communication describes the requirements for this system catalyzed by crude fractions of E. coli. MATERIALS AND METHODS Bacterial and Phage Strains. E. coli and bacteriophage qX174 used and their sources were: E. coli H560 (pol Al, Su+). All other strains were Su-, including BT1029 (pol A1, dnaBts), BT1026 (polAi, dnaEts), and BT1040 (polAj, dnaEts) (Dr. J. Wechsler); E. coli LD332 (dnaCt) (Dr. L. Dumas); E. coli D92 (rep38), a derivative of HF4704 (OXs), and NY73 OX5, a derivative of NY73 (polAl, dnaGts) (Dr. D. Denhardt); E. coli H514 (Dr. R. Knippers); kX174 wild type and kX174 am3 (lysis-, gene E) (Dr. R. L. Sinsheimer), and amN14 (gene A) and amH90 (gene A) (Dr. M. Hayashi). Preparation of DNA and Proteins. Uninfected E. coli was grown and collected; crude ammonium sulfate fractions (also called receptor fractions) were prepared as described (3). The E. coli gene products dnaB (3), dnaC(D) (3), dnaE (15), and dnaG (3) were purified as described. OX174 [3H]RFI and qX174 [3H]RFII were prepared from E. coli HF4704 infected with OX174 am3 in the presence or absence of 30 gig/ml of chloramphenicol (16-18). Abbreviations: RF, replicative form of OX DNA, double-stranded DNA of circular form; RFII, double-stranded DNA of circular replicative form with a discontinuity in at least one strand; ts, temperature-sensitive.
RESULTS Properties of Conversion of OX RF to Progeny RF In Vitro. Crude fractions from 4X174 infected E. coli incorporated dTMP into an acid-insoluble, alkali-resistant, RNase-resistant, DNase-sensitive product when supplemented with crude fractions from uninfected E. coli*. The reaction depended on added OX RFI DNA, provided small amounts of fraction II was used (Table 1, Fig. 1); omission of ATP, dNTPs, or Mg++ also abolished activity. The optimum concentrations of ATP and Mg++ required were approximately 1 mM and 10 mM, respectively. N-Ethylmaleimide abolished DNA synthesis, whereas rifampicin had no effect. Nalidixic acid and novobiocin, specific inhibitors of bacterial DNA synthesis (21, 22), markedly inhibited DNA synthesis (Table 1, Fig. iD); in contrast, formation of OX RFII in vitro from X174 DNA was insensitive to these drugs (data not shown). Replication of OX RFI was inhibited 50% by 50 mM KCI, 15% sucrose (final), or 8.5% glycerol (final). The rate of synthesis was proportional to the amount of fraction II and template DNA added (Fig. 1A, B, and C). Fraction II prepared from cells grown and infected at 300 resulted in higher activity by exogenously added XX RFI with *
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The ammonium sulfate fraction was added as a source of proteins present in uninfected E. coli involved in OX replication.
1888
Biochemistry:
Sumida-Yasumoto et al.
Froc. Natl. Acad. Sci. USA 7.3 (1976)
Table 1. Requirements for synthesis of PX174 progeny RF in vitro Additions
Complete Omit OX RFI Omit ammonium sulfate fraction Omit fraction II Omit ATP or omit Mg++ or omit dATP, dGTP, dCTP Complete + N-ethylmaleimide (4 mM) Complete + rifampicin (10 jg/ml) Complete + nalidixic acid
(240 Ag/ml)
_
Incorporation of dTMP (pmol/40 min) 374 21 6 6
0.
,XRFI
~0
no0 150
-
2
100
0
aL
50&
0 0 2 Fraction 1 (added (MIL)
2
0
0.
400
E
_
z E
0
3
j 200 B
0-
a)
2-4
C T0
a.
C
I-M
30 )°-
>
41
Complete + novobiocin
(240,og/ml)
30e-
10 80
-D
6
0
2C )o 38
Conditions were as described in Materials and Methods, with E. coli H560 ammonium sulfate fraction (0.19 mg of protein) and fraction II (23 jig of protein). Fraction II was prepared from E. coli H560 cells infected with XX am3 phage for 30 min in which cells were grown and infected at 300. Fraction II was prepared as follows: infected frozen cells (equivalent to 0.8 g of cells) from 2 liters of medium were thawed and diluted to 5.4 ml with 10% sucrose containing 50 mM Tris.HCl (pH 7.5). The suspension was then successively treated with 6 j1 of 1 M dithiothreitol, 0.24 ml of 0.5 M EDTA, 0.3 ml of lysozyme (4 mg/ml in 50 mM Tris-HCl, pH 8.0), and 60 ,l of 10% Brij 58 (Sigma). The mixture was incubated at 00 for 25 min and centrifuged for 30 min at 50,000 rpm (Beckman 65 rotor) at 20. The supernatant was adjusted to 45% with liquid ammonium sulfate at 00 (saturated at 00; neutralized with NH40H) added over a 10-min period. After 20 min at 00, the mixture was centrifuged for 15 min at 17,000 rpm (Sorvall SS34 rotor) and the precipitate suspended in 3 ml of buffer A (50 mM Tris.HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol). This fraction (3 ml) was applied to a DEAE-cellulose 52 column (1 x 4 cm) equilibrated with buffer A and eluted with 8 ml of buffer A + 0.25 M NaCl. The flowthrough and 0.25 M NaCl eluate were pooled and adjusted to 45% with liquid ammonium sulfate at 00 as above. After 20 min, the precipitate obtained as above was suspended in 0.3 ml of buffer B (10% sucrose, 10 mM Tris.HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol) followed by dialysis for 1 hr against four changes of 250 ml of buffer B at 00 (fraction II). Fraction II could be stored for 1 day at 00 or at -.80° for 1 month without loss of 4X RF synthetic activity. Repeated freezing and thawing of fraction II destroyed its activity.
small amounts of protein than extracts from cells infected at
02
cE
IC
0
0Iol
I
I
4*0
2
~\
Novobiocin
N ldixic acid
O
.
aid
0 120 240 360 Inhibitor added (pg/ml)
1 2 *6XRFI added(nmol)
FIG. 1. Effect of fraction II, XX RFI, nalidixic acid, and novobiocin on synthesis of .0X progeny RF. Reactions were done as in Materials and Methods, except that fraction II or AX RFI was varied in panels A, B, and C; in panel D, nalidixic acid or novobiocin was added as indicated. Fraction II was prepared as in the legend of Table 1 from E. coli H560 infected with XX am3. (A) fraction II (38 mg/ml of protein) was prepared from cells grown and infected (30 min) at 300, and 1 nmol of OX RFI was added where indicated. (B) Fraction 11 (18 mg/ml of protein) was prepared from cells grown and infected (20 min) at 350, and 1 nmol of XX RFI was added where indicated. (C) Fraction II prepared as described in A (23 jg) (-) or in B (54 jg) (0) was added where indicated. (D) Fraction II (23 jug), prepared as in A, and 1.8 nmol XX RFI were used; 308 pmol of [a-32P]dTMP corresponded to 100% activity. a mixture of 4X RFI and RFII (from cells infected with XX arm3
for 25 min at 350). In the absence of exogenously added kX RFI, a small amount of DNA synthesis was observed (Table 1, Fig. 1A and B); 75% of this reaction was due to conversion of 4tX174 DNA to RFII (measured by product characterization and resistance to novobiocin and nalidixic acid). To date, no DNA other than OX174 or RFI supports DNA synthesis in the
350. Fraction II from uninfected E. coli or from cells infected
with OX wild type or am3 (10-6 min of infection at 350) in the presence of 30 ug/ml of chloramphenicol (to prevent 4X174 DNA synthesis and cell lysis) was inactive for OX RFI-dependent DNA synthesis. Under standard conditions, DNA synthesis plateaued after incorporation of about 400 pmol of dTMP into DNA (with 1.8 nmol of OX RFI). The time required for maximum activity depended on the quantity of fraction II (Figs. 1C and 2). With 2 Al of fraction II (76 ,ug of protein), the reaction was linear for 50 min (Fig. 2) and showed no product degradation upon further incubation (total of 70 min of incubation). With smaller amounts of fraction II (7.6-22.8 ,ug of protein), sigmoidal kinetics were observed as well as some degradation of the product (20-30%) with further incubation after incorporation of dTMP ceased. Fraction II contained endogenous DNA that was exclusively YX DINA. In cnrde ammonium sulfate fractions, approximately 35% of the DNA was present as 4X174 DNA and the rest was
-3 06 2 (7-)
/A
0
06.
0
0.
(3g
C
I
0
10
20
30
40
5 70
Time of incubation (min)
FIG. 2. Kinetics of synthesis of OX progeny RF in vitro. Reaction mixtures were as described in Materials and Methods with variable amounts of fraction II (see Table 1): 76 gg (0); 23 jg (0); or 7.6 jg (a) of fraction II and 1.8 nmol AX RFI were used. (X) Indicates no addition of XX RFI with 23 gg of fraction II.
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Sunlida-Yasumoto et al. Table 2. Template specificity for DNA synthesis Incorporation of dTMP (pmol/40 min)
Template DNA added
,OX RFI pX174 DNA pXRFII ColEl PM 2 NativeT3 Omit DNA
226 141 19 8 18 7 21
(1.8) (1.8) (0.9) (1.8) (0.6) (0.8)
Reaction mixtures were as described in Table 1, .except XX RFI was replaced where indicated with other DNAs and 0.18 mg of protein of ammonium sulfate fraction was used. The numbers in parentheses indicate the amount of DNA (in nmol of nucleotide) added. Colicin E1 was prepared as described (20), while PM 2 and native T3 DNAs were gifts from Drs. S. Wallace and U. Maitra, respectively.
system used (Table 2); circular DNAs, such as PM2, colicin El, or OX RFII, and linear native T3 DNA were inactive.
Characterization of XX Progeny RF. DNA synthesized in vitro with [3H]RFI was analyzed by zonal sedimentation in neutral and alkaline sucrose (Fig. 3). In neutral gradients the DNA product sedimented as expected of RFI and RFII. The amount of these two forms varied, and the ratio of RFI to RFII increased with incubation: 40 and 60% of the product was RFI and RFII, respectively, after 10 mMi of incubation; after 40 mm of incubation the products contained 70 and 30% RFI and RFII, respectively. Similar results were obtained when products were analyzed by alkaline sucrose gradient centrifugation (Fig. 3). These results suggest that the initial product of the reaction is RFII, which is then converted to RFI. In alkaline gradients, single-stranded DNA sedimented more heterogeneously than the reference kX174 DNA marker (Fig. 3B). DNA longer than one unit length was detected among the products formed after 10 min of incubation, suggesting but not proving that OX RFI A
F)
1889
may replicate via a rolling circle structure; small fragments of DNA were detected in alkaline gradients (10-15% of the DNA
product); no such products were observed in neutral gradients. Newly formed OX RFI had the same superhelical density as that of the template RFI, as measured by isopycnic banding in propidium diiodide-neutral CsCl (data not shown). Thus, the characteristics of the product suggest that the reaction catalyzed by the cell-free system results in the replication of OX RF. To demonstrate that RFI replication occurred semiconservatively, we synthesized DNA in vitro with [3H]dATP and 5-bromodeoxyuridine 5'-triphosphate (BrdUTP) in place of dTTP, and the product was subjected to neutral sucrose gradient and CsCl equilibrium gradient centrifugation. Analysis of the product formed after 40 min (Fig. 4A) showed that 20 and 80% of BrdUrd-labeled DNA sedimented in neutral sucrose as RFI and RFII forms, respectively. These products sedimented slightly faster than RFI and RFII containing only dTMP. More RFII than RFI was always observed in products of density-labeled DNA compared to products formed without BrdUTP. When analyzed by neutral CsCl density centrifugation (Fig. 4B), over 65% of newly synthesized [32P]DNA and 20% of template [3H]DNA banded at the density of hybrid molecules (H-L). Approximately 20% of 32P-labeled product banded as expected of fully heavy (H-H) DNA molecules. In other experiments, in which products were subjected to isopycnic banding in propidium diiodide-neutral CsCl, both RFI and RFII forms were mixtures of H-H and H-L forms (data not shown). Involvement of E. coli Proteins and OX Gene A Product in RF Replication. It has been established that E. coli proteins dnaB, C(D), E, and C and the rep gene (8-13) are involved in OX RF replication. Gene A, coded for by the XX genome, appears to be the only phage function essential for RF duplication (14). The roles of these proteins were examined using various mutants as receptor and fraction II for RF replication in vitro (Table 3). dnaC(D): Reactions containing fraction II isolated from E. coli H560 infected with OX am3 and ammonium sulfate frac-
neutral gradient (40 Min)
4
0 4
II
2
2
I
10 0.6
20
a.
40
30
C alkaline gradient (20 min)
I
N
PI)
20%
I I0
5
10
20
30
4
FRACTION NUMBER FIG. 3. Sucrose gradient analysis of DNA synthesized in vitro. DNA was synthesized with (a-32P]dTTP (400 cpm/pmol), OX [3H]RFI (6 cpm/pmol), and fraction II (54 ,ug) (see legend of Fig. 1B). Incubations were at 30° for 10 (B), 20 (C), and 40 (A and D) min. DNA was analyzed by neutral (A) and alkaline sucrose gradient centrifugation (B, C, and D). Samples were layered onto 5-ml, linear 5-20% neutral or alkaline sucrose gradients containing 10 mM Tris-HCl (pH 8.0), 1 M NaCl, 10 mM EDTA, and 0.05% Sarkosyl (or 0.25 M NaOH for alkaline gradients), and sedimented for 15 hr at 25,000 rpm or 2 hr at 50,000 rpm, respectively, in the SW-50.1 rotor at 4°. Fractions were collected from the bottom of the tube, and acid-insoluble radioactivity was determined. A reference OX174 [3H]DNA marker was run in parallel gradient (X) with XX [3H]RFI; arrows in C and D indicate the position of reference kX174 DNA.
1890
Biochemistry: Sumida-Yasumoto et al.
Froc. Nati. Acad. Sci. USA 73 (1976)
Table 3. Involvement of E. coli proteins and
OX gene A product in OX RF synthesis
Source of E. coli extract Fraction II and phage used for infection BT1029 (OXwt) BT1029 (oXwt)
H560 (4Xam3) H560 (oXam3) NY73 OXS (OXwt) NY73 oXS (oXwt) D92 (rep-) (OXwt) D92 (rep-) (OXwt) H514 (pXwt)
H5i4 (OXH90) H514 (OXH90)
Ammonium sulfate fraction (uninfected)
BT1029 BT1029 LD332 LD332
NY73OXS
NY734XS D92 (rep-) H560 (rep+) H514 H514 H514
Incorporation of dTMP (pmol/40 min) Purified gene product added
Without heat treatment
After heat treatment
None dnaB None dnaC None dnaG None None None None OX gene A
106 192 28 568 461 460 5 207 121 9 120
41 219 57 250
Fraction II was prepared as described in the legend of Table 1. E. coli strains BT1029 and NY73 OXs were infected with OX wild-type phage
(OXwt) for 30 and 45 min, respectively, and grown and infected at 300. Strains H560 and D92 were infected for 20 min with OX am3 and OX wild type, respectively; H514 was infected for 20 min with OX wild type or for 30 min with OXH90 (or OXN14) at 35°. Reactions were as described in Materials and Methods, with fraction II protein as follows: H560 (28 ,g), BT1029 (67 Ag), NY73 OXs (24 ,g), D92 (60 ,ug), H514 infected with OX wild type (57 Mg), and H514 infected with 4XH90 (30 Mg). Where indicated, fraction II and uninfected ammonium sulfate fractions were heated (10-20 min at 38° or 5-10 min at 41°) immediately prior to use. Purified preparations of dna gene products (&.05-0.1 unit) were added where indicated. In experiments with heated extracts involving dnaB and dnaG ts extracts, incorporation of dTMP in the presence of nalidixic acid was 20 and 25 pmol, respectively, in the presence or absence of added gene products. In experiments with dnaC, incorporation with or without added dnaC gene product was 38 and 29 pmol, respectively, in the presence of nalidixic acid.
tions from LD332 were inactive in RF synthesis without prior heat treatmentt. Addition of purified dnaC(D) gene product stimulated the reaction 20-fold (Table 3); addition of other gene products to reaction mixtures derived from these strains did not stimulate RF replication. dnaB and dnaG: Reaction mixtures containing fractions from E. coli strains BT1029 (danBb) and NY73 40Xs (dnaGls) were thermolabile; addition of purified preparations of dnaB and dnaC gene products to suitable extracts after heat treatment stimulated RF replication about 5-fold in each case. These results suggest that both dnaB and dnaG gene products are involved in progeny RF synthesis. In progeny RF synthesis, the requirement for dnaBl and dnaG were satisfied at lower concentrations than that required for conversion of OX174 DNA to RFII (data not shown). dnaE: RFI replication was stimulated 50% by addition of purified DNA polymerase III (dnaE gene product) t6 the system prior to heating of dnaEb fractions (derived from BT1026 and BT1040). Heat treatment of different dnaEts extracts reduced fX RF replication. Addition of purified DNA polymerase III did not restore RF replication (data not shown). At present, the role of DNA polymerase III in RF replication must await further studies. rep gene: Protein fractions from E. coli strain D92 (rep-) were inactive in RF replication but were active in converting. ,X174 DNA to RFII (data not shown). These results suggest that the rep gene is required for RF replication but not for parental RF synthesis. Mixing experiments suggested that this system was inactive due to a lack of the rep gene product. The combination of fraction II from D92 (rep-) and receptor from H560 sulfate fractions prepared from E. coil strain LD332 (dnaCts) lacked detectable dnaC activity. The small amount of fraction II protein added was deficient in dnaC gene product. Thus, fractions from these two strains contained no dnaC gene product.
t Ammonium
(rep+) was active. The alternate combination was also active (data not shown). 4X174 gene A: Fraction II prepared from E. coli H514 infected with OX amH90 or amN14 (data not shown) was inactive in RF replication when combined with the receptor from the same strain. The addition of purified gene A productt stimulated the reaction 10-fold. In all experiments reported above, synthesis of OX RF DNA stimulated by different gene products was (90%) sensitive to novobiocin and nalidixic acid. DISCUSSION Extracts prepared from OX-infected E. coli plus extracts from uninfected cells catalyze semiconservative OX RF replication with exogenous XX RFI as template. Maximal activity required Mg++, four dNTPs, ATP, and k6X gene A product in addition to the host gene products, dnaB, dnaC(D), dnaG, and rep gene. The reaction is rifampicin-resistant but sensitive to nalidixic acid and novobiocin. These results are consistent with observations in vivo; however, the dnaE requirement observed in vivo (9) could not be satisfied in vitro by DNA polymerase III. In some experiments (data not presented) DNA synthesis was enhanced by addition of the four NTPs, but these observations were not reproducible. At present, the role of NTPs in OX RF DNA synthesis is unclear. The products formed by this system are OX RFI and OX RFII. After 10 min of synthesis, 60% of DNA synthesized with extracts derived from pol A strains was OX RFII, which after further incubation was converted to RFI. OX RFI synthesized in vitro appears to have the same superhelical density as 4X RFI isolated in vivo. These results contrast with those obtained with t The gene A product used was purified 2000-fold by Drs. Joh-e Ikeda and A. Yudelevich of this Department. In addition, the RF replication system is capable of catalyzing net synthesis of DNA.
Proc. Natl. Acad. Sct. USA 73 (1976)
Biochemistry: Sumida-Yasumoto et al.
1891
Neutral sucrose gradient
A 2
*XRFI *XRF x
_C,2 0I
I
,
a.
'A
2 I
I?
I
x
a-
CQ Fe)
20 30 40 10 20 30 40 50 60 10 NUMBER FRACTION FIG. 4. Neutral sucrose gradient and buoyant density sedimentation analyses of BrdUrd-labeled XX DNA synthesized in vitro. DNA was synthesized with [a-32P]dATP (400 cpm/pmol), XX [3HJRFI (6 cpm/pmol), 40guM BrdUTP (in place of dTTP), and fraction II (54 lug) prepared as described in the legend of Fig. 1B. DNA was subjected to neutral sucrose gradient (A) as described in the legend of Fig. 3 and density sedimentation in neutral CsCl (B). (A) Arrows indicate the positions of reference OX [3H]RFI and [3H]RFII markers. (B) For neutral CsCl analysis, the volume was adjusted to 3 ml with 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM NaCl to which 3.7 g of CsCl was added. The mixture was sedimented for 64 hr at 35,000 rpm in a SW-50.1 rotor at 200. Fractions were collected from the bottom of the tube, and acid-insoluble material was measured. Arrows indicate the positions expected for fully heavy (H-H), half-heavy (H-L), and completely light (L-L) OX RFI DNA. vitro system in which DNA polymerase I plus DNA ligase convert M13 or OX RFII to RFI (23, 6). The latter products an in
possessed higher buoyant densities, as measured by isopycnic banding in ethidium bromide-CsCl, than the corresponding forms in vivo. We have devised complementation assays for various proteins essential in OX RFI replication. This technique, already used to isolate proteins involved in the conversion of kX174 DNA to RFII (3), has permitted us to isolate the OX gene A proteint as well as the rep give (unpublished observation). A cell-free system has been developed by modifying the procedure described in Materials and Methods in which OX RFI-dependent OX 174 DNA synthesis occurs (unpublished observation). Thus, single-stranded circular phage DNA can now be replicated through the three stages described in the introduction with cell-free preparations. We are indebted to Drs. J. Wecshler, L. Dumas, D. Denhardt, R. L. Sinsheimer, H. Hayashi, and R. Knippers for supplying E. coli strains and mutants of phage OX174. We thank Dr. S. Wickner for many suggestions. This work was supported by the National Institutes of Health, the National Science Foundation and The American Cancer Society. 1. Wickner, W. T., Brutlag, D., Schekman, R. & Kornberg, A. (1972) Proc. Natl. Acad. Sci. USA 69,965-969. 2. Wickner, R. B., Wright, M., Wickner, S. & Hurwitz, J. (1972) Proc. Natl. Acad. Sci. USA 69,3233-3237. 3. Wickner, S., Wright, M., Berkower, I. & Hurwitz, J. (1974) in DNA Replication, ed. Wickner, R. B. (Marcel Dekker, Inc., New York), pp. 195-215.
4.
Wickner, S. & Hurwitz, J. (1976) Proc. Natl. Acad. Sci. USA 73,
1053-1057. 5. Wickner, S. & Hurwitz, J. (1974) Proc. Natl. Aced. Sci. USA 71, 4120-4124. 6. Schekman, R., Weiner, A. & Kornberg, A. (1974) Science 186, 987-993. 7. Schekman, R., Weiner, H. J., Weiner, A. & Kornberg, A. (1974) 8.
9. 10. 11. 12. 13. 14.
J. Biol. Chem. 250,5859-5865. Kranias, E. G. & Dumas, L. B. (1974) J. Virol. 13, 146-154. Dumas, L. B. & Miller, C. A. (1973) J. Virol. 11, 848-855. McFadden, G. & Denhardt, D. T. (9174) J. Virol. 14,1070-1075. Truitt, C. L. & Walker, J. R. (1974) Biochem. Biophys. Res. Commun. 61,1036-1042. Taketo, A. (1975) Mol. Gen. Genet. 139,285-291. Denhardt, D. T., Dressler, D. H. & Hathaway, A. (1967) Proc. Nati. Acad. Sci. USA 57,813-820. Lindquist, B. H. & Sinsheimer, R. L. (1967) J. Mol. Biol. 30,
69-80. 15. Hurwitz, J.- & Wickner, S. (1974) Proc. Natl. Acad. Sci. USA 71, 6-10. 16. Sclair, M., Edgell, M. H. & Hutchinson, C. A. (1973) J. Virol. 11, 378-85. 17. Vapnek, D. & Rupp, W. D. (1971) J. Mol. Biol. 60,413. 18. Komano, T. & Sinsheimer, R. L. (1968) Biochim. Biophys. Acta 155,295. 19. Francke, B. & Ray, D. S. (1971) Virology 44, 168. 20. Sakakibara, Y. & Tomizawa, J. (1974) Proc. Natl. Acad. Sci. USA
71,802-806. 21. Goss, W. A., Deitz, W. H. & Cook, T. M. (1965) J. Bacteriol. 89, 1068-1074. 22. Staudenbauer, W. L. (1975) J. Mol. Biol. 96,201-205. 23. Geider, K. & Kornberg, A. (1974) J. Biol. Chem. 249,3999-4005.
Biochemistry
DNA synthesis in vitro dependent upon kX174 replicative form I DNA (OX A gene product/DNA replication) CHIKAKO SUMIDA-YASUMOTO, ARTURO YUDELEVICH, AND JERARD HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461
Contributed by Jerard Hurwitz, March 17,1976
Cells were grown and infected with phage at 300 for E. coli dna temperature-sensitive (ts) mutants or 350 for other E. coli strains, unless noted. Cells were grown to an OD65o of 0.3 in medium supplemented with 4 jig/ml of thymine (19), infected with OX wild type or mutants with 5-10 phages/bacterium, and incubated for 5 min without shaking to allow adsorption. CaCd2 was then added (final concentration 1 mM) and infection continued as indicated in figure and legends. Cells were collected and frozen (3) and stored at -80° for at least 1 month without loss of XX RF synthetic activity. Preparation of fraction II is detailed in the legend of Table 1. Assay of DNA Synthesis. Standard reaction mixtures (0.05 ml) contained, in order of addition, 20 mM Tris.HCl (pH 7.5), 10 mM MgCl2, 4 mM dithiothreitol, 1 mM ATP, 1 mM NAD+, 40 AM each of dATP, dCTP, dGTP, and [a-32P]dTTP (200-700 cpm/pmol), 100MuM each of CTP, UTP, and GTP, 1.8 nmol of OX [3H]RFI, (6 cpm/pmol), 5 Al of ammonium sulfate fraction from uninfected cells (protein as indicated), and fraction II (prepared from OX-infected cells described below). Reaction mixtures were incubated for 40 min at 300 (unless specified), and acid-insoluble radioactivity was determined (3). When products were characterized by sedimentation analyses, samples were treated either as described by Sakakibara and Tomizawa (20) or by addition of EDTA (24 mM) and Sarkosyl (final 2%) followed by incubation at 420 for 10 min.
Extracts of Escherichia coli strains infected ABSTRACT with bacteriophage 4X174 catalyze DNA synthesis dependent on double-stranded, circular kX174 replicative form I (OX RFI) by a semiconservative process. The reaction required Mg++, ATP, all four dNTP, and exogenous XX RFI DNA as teaplate and yielded OX RFI and OX RFII. The reaction was inhibited by nalidixic acid and novobiocin but not by rifampicin. DNA synthesis required the OX174 gene A product and E. coli gene products dnaB, dnaC(D), dnaG, and rep.
Replication of /X174 single-stranded, circular DNA occurs in three stages: synthesis of (a) parental replicative form (RF), (b) progeny RF, and (c) progeny single-stranded, circular DNA. In stage a, parental RF synthesis depends solely upon host proteins, and a cell-free system catalyzing this reaction has been developed (1, 2). Complementation assays with this system allowed the isolation of Escherichia coli dnaB, C(D), G, and Z gene products (3, 4) and other proteins required for conversion of OX174 DNA to RFII (5-7). This system is specific for OX174 DNA and is inactive with duplex OXRF forms. In avo, progeny RF and/or progeny qX174 DNA synthesis also depends upon E. coli dnaB (8), dnaC(D) (9), dnaE (9), dnaG (10), dnaZ (11, 12), and rep gene (13) products in addition to the kX174 gene A product (14). To study the mechanism of double-stranded DNA replication, we have developed a cell-free system dependent upon added OX RFI DNtA. This communication describes the requirements for this system catalyzed by crude fractions of E. coli. MATERIALS AND METHODS Bacterial and Phage Strains. E. coli and bacteriophage qX174 used and their sources were: E. coli H560 (pol Al, Su+). All other strains were Su-, including BT1029 (pol A1, dnaBts), BT1026 (polAi, dnaEts), and BT1040 (polAj, dnaEts) (Dr. J. Wechsler); E. coli LD332 (dnaCt) (Dr. L. Dumas); E. coli D92 (rep38), a derivative of HF4704 (OXs), and NY73 OX5, a derivative of NY73 (polAl, dnaGts) (Dr. D. Denhardt); E. coli H514 (Dr. R. Knippers); kX174 wild type and kX174 am3 (lysis-, gene E) (Dr. R. L. Sinsheimer), and amN14 (gene A) and amH90 (gene A) (Dr. M. Hayashi). Preparation of DNA and Proteins. Uninfected E. coli was grown and collected; crude ammonium sulfate fractions (also called receptor fractions) were prepared as described (3). The E. coli gene products dnaB (3), dnaC(D) (3), dnaE (15), and dnaG (3) were purified as described. OX174 [3H]RFI and qX174 [3H]RFII were prepared from E. coli HF4704 infected with OX174 am3 in the presence or absence of 30 gig/ml of chloramphenicol (16-18). Abbreviations: RF, replicative form of OX DNA, double-stranded DNA of circular form; RFII, double-stranded DNA of circular replicative form with a discontinuity in at least one strand; ts, temperature-sensitive.
RESULTS Properties of Conversion of OX RF to Progeny RF In Vitro. Crude fractions from 4X174 infected E. coli incorporated dTMP into an acid-insoluble, alkali-resistant, RNase-resistant, DNase-sensitive product when supplemented with crude fractions from uninfected E. coli*. The reaction depended on added OX RFI DNA, provided small amounts of fraction II was used (Table 1, Fig. 1); omission of ATP, dNTPs, or Mg++ also abolished activity. The optimum concentrations of ATP and Mg++ required were approximately 1 mM and 10 mM, respectively. N-Ethylmaleimide abolished DNA synthesis, whereas rifampicin had no effect. Nalidixic acid and novobiocin, specific inhibitors of bacterial DNA synthesis (21, 22), markedly inhibited DNA synthesis (Table 1, Fig. iD); in contrast, formation of OX RFII in vitro from X174 DNA was insensitive to these drugs (data not shown). Replication of OX RFI was inhibited 50% by 50 mM KCI, 15% sucrose (final), or 8.5% glycerol (final). The rate of synthesis was proportional to the amount of fraction II and template DNA added (Fig. 1A, B, and C). Fraction II prepared from cells grown and infected at 300 resulted in higher activity by exogenously added XX RFI with *
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The ammonium sulfate fraction was added as a source of proteins present in uninfected E. coli involved in OX replication.
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Biochemistry:
Sumida-Yasumoto et al.
Froc. Natl. Acad. Sci. USA 7.3 (1976)
Table 1. Requirements for synthesis of PX174 progeny RF in vitro Additions
Complete Omit OX RFI Omit ammonium sulfate fraction Omit fraction II Omit ATP or omit Mg++ or omit dATP, dGTP, dCTP Complete + N-ethylmaleimide (4 mM) Complete + rifampicin (10 jg/ml) Complete + nalidixic acid
(240 Ag/ml)
_
Incorporation of dTMP (pmol/40 min) 374 21 6 6
0.
,XRFI
~0
no0 150
-
2
100
0
aL
50&
0 0 2 Fraction 1 (added (MIL)
2
0
0.
400
E
_
z E
0
3
j 200 B
0-
a)
2-4
C T0
a.
C
I-M
30 )°-
>
41
Complete + novobiocin
(240,og/ml)
30e-
10 80
-D
6
0
2C )o 38
Conditions were as described in Materials and Methods, with E. coli H560 ammonium sulfate fraction (0.19 mg of protein) and fraction II (23 jig of protein). Fraction II was prepared from E. coli H560 cells infected with XX am3 phage for 30 min in which cells were grown and infected at 300. Fraction II was prepared as follows: infected frozen cells (equivalent to 0.8 g of cells) from 2 liters of medium were thawed and diluted to 5.4 ml with 10% sucrose containing 50 mM Tris.HCl (pH 7.5). The suspension was then successively treated with 6 j1 of 1 M dithiothreitol, 0.24 ml of 0.5 M EDTA, 0.3 ml of lysozyme (4 mg/ml in 50 mM Tris-HCl, pH 8.0), and 60 ,l of 10% Brij 58 (Sigma). The mixture was incubated at 00 for 25 min and centrifuged for 30 min at 50,000 rpm (Beckman 65 rotor) at 20. The supernatant was adjusted to 45% with liquid ammonium sulfate at 00 (saturated at 00; neutralized with NH40H) added over a 10-min period. After 20 min at 00, the mixture was centrifuged for 15 min at 17,000 rpm (Sorvall SS34 rotor) and the precipitate suspended in 3 ml of buffer A (50 mM Tris.HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol). This fraction (3 ml) was applied to a DEAE-cellulose 52 column (1 x 4 cm) equilibrated with buffer A and eluted with 8 ml of buffer A + 0.25 M NaCl. The flowthrough and 0.25 M NaCl eluate were pooled and adjusted to 45% with liquid ammonium sulfate at 00 as above. After 20 min, the precipitate obtained as above was suspended in 0.3 ml of buffer B (10% sucrose, 10 mM Tris.HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol) followed by dialysis for 1 hr against four changes of 250 ml of buffer B at 00 (fraction II). Fraction II could be stored for 1 day at 00 or at -.80° for 1 month without loss of 4X RF synthetic activity. Repeated freezing and thawing of fraction II destroyed its activity.
small amounts of protein than extracts from cells infected at
02
cE
IC
0
0Iol
I
I
4*0
2
~\
Novobiocin
N ldixic acid
O
.
aid
0 120 240 360 Inhibitor added (pg/ml)
1 2 *6XRFI added(nmol)
FIG. 1. Effect of fraction II, XX RFI, nalidixic acid, and novobiocin on synthesis of .0X progeny RF. Reactions were done as in Materials and Methods, except that fraction II or AX RFI was varied in panels A, B, and C; in panel D, nalidixic acid or novobiocin was added as indicated. Fraction II was prepared as in the legend of Table 1 from E. coli H560 infected with XX am3. (A) fraction II (38 mg/ml of protein) was prepared from cells grown and infected (30 min) at 300, and 1 nmol of OX RFI was added where indicated. (B) Fraction 11 (18 mg/ml of protein) was prepared from cells grown and infected (20 min) at 350, and 1 nmol of XX RFI was added where indicated. (C) Fraction II prepared as described in A (23 jg) (-) or in B (54 jg) (0) was added where indicated. (D) Fraction II (23 jug), prepared as in A, and 1.8 nmol XX RFI were used; 308 pmol of [a-32P]dTMP corresponded to 100% activity. a mixture of 4X RFI and RFII (from cells infected with XX arm3
for 25 min at 350). In the absence of exogenously added kX RFI, a small amount of DNA synthesis was observed (Table 1, Fig. 1A and B); 75% of this reaction was due to conversion of 4tX174 DNA to RFII (measured by product characterization and resistance to novobiocin and nalidixic acid). To date, no DNA other than OX174 or RFI supports DNA synthesis in the
350. Fraction II from uninfected E. coli or from cells infected
with OX wild type or am3 (10-6 min of infection at 350) in the presence of 30 ug/ml of chloramphenicol (to prevent 4X174 DNA synthesis and cell lysis) was inactive for OX RFI-dependent DNA synthesis. Under standard conditions, DNA synthesis plateaued after incorporation of about 400 pmol of dTMP into DNA (with 1.8 nmol of OX RFI). The time required for maximum activity depended on the quantity of fraction II (Figs. 1C and 2). With 2 Al of fraction II (76 ,ug of protein), the reaction was linear for 50 min (Fig. 2) and showed no product degradation upon further incubation (total of 70 min of incubation). With smaller amounts of fraction II (7.6-22.8 ,ug of protein), sigmoidal kinetics were observed as well as some degradation of the product (20-30%) with further incubation after incorporation of dTMP ceased. Fraction II contained endogenous DNA that was exclusively YX DINA. In cnrde ammonium sulfate fractions, approximately 35% of the DNA was present as 4X174 DNA and the rest was
-3 06 2 (7-)
/A
0
06.
0
0.
(3g
C
I
0
10
20
30
40
5 70
Time of incubation (min)
FIG. 2. Kinetics of synthesis of OX progeny RF in vitro. Reaction mixtures were as described in Materials and Methods with variable amounts of fraction II (see Table 1): 76 gg (0); 23 jg (0); or 7.6 jg (a) of fraction II and 1.8 nmol AX RFI were used. (X) Indicates no addition of XX RFI with 23 gg of fraction II.
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Sunlida-Yasumoto et al. Table 2. Template specificity for DNA synthesis Incorporation of dTMP (pmol/40 min)
Template DNA added
,OX RFI pX174 DNA pXRFII ColEl PM 2 NativeT3 Omit DNA
226 141 19 8 18 7 21
(1.8) (1.8) (0.9) (1.8) (0.6) (0.8)
Reaction mixtures were as described in Table 1, .except XX RFI was replaced where indicated with other DNAs and 0.18 mg of protein of ammonium sulfate fraction was used. The numbers in parentheses indicate the amount of DNA (in nmol of nucleotide) added. Colicin E1 was prepared as described (20), while PM 2 and native T3 DNAs were gifts from Drs. S. Wallace and U. Maitra, respectively.
system used (Table 2); circular DNAs, such as PM2, colicin El, or OX RFII, and linear native T3 DNA were inactive.
Characterization of XX Progeny RF. DNA synthesized in vitro with [3H]RFI was analyzed by zonal sedimentation in neutral and alkaline sucrose (Fig. 3). In neutral gradients the DNA product sedimented as expected of RFI and RFII. The amount of these two forms varied, and the ratio of RFI to RFII increased with incubation: 40 and 60% of the product was RFI and RFII, respectively, after 10 mMi of incubation; after 40 mm of incubation the products contained 70 and 30% RFI and RFII, respectively. Similar results were obtained when products were analyzed by alkaline sucrose gradient centrifugation (Fig. 3). These results suggest that the initial product of the reaction is RFII, which is then converted to RFI. In alkaline gradients, single-stranded DNA sedimented more heterogeneously than the reference kX174 DNA marker (Fig. 3B). DNA longer than one unit length was detected among the products formed after 10 min of incubation, suggesting but not proving that OX RFI A
F)
1889
may replicate via a rolling circle structure; small fragments of DNA were detected in alkaline gradients (10-15% of the DNA
product); no such products were observed in neutral gradients. Newly formed OX RFI had the same superhelical density as that of the template RFI, as measured by isopycnic banding in propidium diiodide-neutral CsCl (data not shown). Thus, the characteristics of the product suggest that the reaction catalyzed by the cell-free system results in the replication of OX RF. To demonstrate that RFI replication occurred semiconservatively, we synthesized DNA in vitro with [3H]dATP and 5-bromodeoxyuridine 5'-triphosphate (BrdUTP) in place of dTTP, and the product was subjected to neutral sucrose gradient and CsCl equilibrium gradient centrifugation. Analysis of the product formed after 40 min (Fig. 4A) showed that 20 and 80% of BrdUrd-labeled DNA sedimented in neutral sucrose as RFI and RFII forms, respectively. These products sedimented slightly faster than RFI and RFII containing only dTMP. More RFII than RFI was always observed in products of density-labeled DNA compared to products formed without BrdUTP. When analyzed by neutral CsCl density centrifugation (Fig. 4B), over 65% of newly synthesized [32P]DNA and 20% of template [3H]DNA banded at the density of hybrid molecules (H-L). Approximately 20% of 32P-labeled product banded as expected of fully heavy (H-H) DNA molecules. In other experiments, in which products were subjected to isopycnic banding in propidium diiodide-neutral CsCl, both RFI and RFII forms were mixtures of H-H and H-L forms (data not shown). Involvement of E. coli Proteins and OX Gene A Product in RF Replication. It has been established that E. coli proteins dnaB, C(D), E, and C and the rep gene (8-13) are involved in OX RF replication. Gene A, coded for by the XX genome, appears to be the only phage function essential for RF duplication (14). The roles of these proteins were examined using various mutants as receptor and fraction II for RF replication in vitro (Table 3). dnaC(D): Reactions containing fraction II isolated from E. coli H560 infected with OX am3 and ammonium sulfate frac-
neutral gradient (40 Min)
4
0 4
II
2
2
I
10 0.6
20
a.
40
30
C alkaline gradient (20 min)
I
N
PI)
20%
I I0
5
10
20
30
4
FRACTION NUMBER FIG. 3. Sucrose gradient analysis of DNA synthesized in vitro. DNA was synthesized with (a-32P]dTTP (400 cpm/pmol), OX [3H]RFI (6 cpm/pmol), and fraction II (54 ,ug) (see legend of Fig. 1B). Incubations were at 30° for 10 (B), 20 (C), and 40 (A and D) min. DNA was analyzed by neutral (A) and alkaline sucrose gradient centrifugation (B, C, and D). Samples were layered onto 5-ml, linear 5-20% neutral or alkaline sucrose gradients containing 10 mM Tris-HCl (pH 8.0), 1 M NaCl, 10 mM EDTA, and 0.05% Sarkosyl (or 0.25 M NaOH for alkaline gradients), and sedimented for 15 hr at 25,000 rpm or 2 hr at 50,000 rpm, respectively, in the SW-50.1 rotor at 4°. Fractions were collected from the bottom of the tube, and acid-insoluble radioactivity was determined. A reference OX174 [3H]DNA marker was run in parallel gradient (X) with XX [3H]RFI; arrows in C and D indicate the position of reference kX174 DNA.
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Biochemistry: Sumida-Yasumoto et al.
Froc. Nati. Acad. Sci. USA 73 (1976)
Table 3. Involvement of E. coli proteins and
OX gene A product in OX RF synthesis
Source of E. coli extract Fraction II and phage used for infection BT1029 (OXwt) BT1029 (oXwt)
H560 (4Xam3) H560 (oXam3) NY73 OXS (OXwt) NY73 oXS (oXwt) D92 (rep-) (OXwt) D92 (rep-) (OXwt) H514 (pXwt)
H5i4 (OXH90) H514 (OXH90)
Ammonium sulfate fraction (uninfected)
BT1029 BT1029 LD332 LD332
NY73OXS
NY734XS D92 (rep-) H560 (rep+) H514 H514 H514
Incorporation of dTMP (pmol/40 min) Purified gene product added
Without heat treatment
After heat treatment
None dnaB None dnaC None dnaG None None None None OX gene A
106 192 28 568 461 460 5 207 121 9 120
41 219 57 250
Fraction II was prepared as described in the legend of Table 1. E. coli strains BT1029 and NY73 OXs were infected with OX wild-type phage
(OXwt) for 30 and 45 min, respectively, and grown and infected at 300. Strains H560 and D92 were infected for 20 min with OX am3 and OX wild type, respectively; H514 was infected for 20 min with OX wild type or for 30 min with OXH90 (or OXN14) at 35°. Reactions were as described in Materials and Methods, with fraction II protein as follows: H560 (28 ,g), BT1029 (67 Ag), NY73 OXs (24 ,g), D92 (60 ,ug), H514 infected with OX wild type (57 Mg), and H514 infected with 4XH90 (30 Mg). Where indicated, fraction II and uninfected ammonium sulfate fractions were heated (10-20 min at 38° or 5-10 min at 41°) immediately prior to use. Purified preparations of dna gene products (&.05-0.1 unit) were added where indicated. In experiments with heated extracts involving dnaB and dnaG ts extracts, incorporation of dTMP in the presence of nalidixic acid was 20 and 25 pmol, respectively, in the presence or absence of added gene products. In experiments with dnaC, incorporation with or without added dnaC gene product was 38 and 29 pmol, respectively, in the presence of nalidixic acid.
tions from LD332 were inactive in RF synthesis without prior heat treatmentt. Addition of purified dnaC(D) gene product stimulated the reaction 20-fold (Table 3); addition of other gene products to reaction mixtures derived from these strains did not stimulate RF replication. dnaB and dnaG: Reaction mixtures containing fractions from E. coli strains BT1029 (danBb) and NY73 40Xs (dnaGls) were thermolabile; addition of purified preparations of dnaB and dnaC gene products to suitable extracts after heat treatment stimulated RF replication about 5-fold in each case. These results suggest that both dnaB and dnaG gene products are involved in progeny RF synthesis. In progeny RF synthesis, the requirement for dnaBl and dnaG were satisfied at lower concentrations than that required for conversion of OX174 DNA to RFII (data not shown). dnaE: RFI replication was stimulated 50% by addition of purified DNA polymerase III (dnaE gene product) t6 the system prior to heating of dnaEb fractions (derived from BT1026 and BT1040). Heat treatment of different dnaEts extracts reduced fX RF replication. Addition of purified DNA polymerase III did not restore RF replication (data not shown). At present, the role of DNA polymerase III in RF replication must await further studies. rep gene: Protein fractions from E. coli strain D92 (rep-) were inactive in RF replication but were active in converting. ,X174 DNA to RFII (data not shown). These results suggest that the rep gene is required for RF replication but not for parental RF synthesis. Mixing experiments suggested that this system was inactive due to a lack of the rep gene product. The combination of fraction II from D92 (rep-) and receptor from H560 sulfate fractions prepared from E. coil strain LD332 (dnaCts) lacked detectable dnaC activity. The small amount of fraction II protein added was deficient in dnaC gene product. Thus, fractions from these two strains contained no dnaC gene product.
t Ammonium
(rep+) was active. The alternate combination was also active (data not shown). 4X174 gene A: Fraction II prepared from E. coli H514 infected with OX amH90 or amN14 (data not shown) was inactive in RF replication when combined with the receptor from the same strain. The addition of purified gene A productt stimulated the reaction 10-fold. In all experiments reported above, synthesis of OX RF DNA stimulated by different gene products was (90%) sensitive to novobiocin and nalidixic acid. DISCUSSION Extracts prepared from OX-infected E. coli plus extracts from uninfected cells catalyze semiconservative OX RF replication with exogenous XX RFI as template. Maximal activity required Mg++, four dNTPs, ATP, and k6X gene A product in addition to the host gene products, dnaB, dnaC(D), dnaG, and rep gene. The reaction is rifampicin-resistant but sensitive to nalidixic acid and novobiocin. These results are consistent with observations in vivo; however, the dnaE requirement observed in vivo (9) could not be satisfied in vitro by DNA polymerase III. In some experiments (data not presented) DNA synthesis was enhanced by addition of the four NTPs, but these observations were not reproducible. At present, the role of NTPs in OX RF DNA synthesis is unclear. The products formed by this system are OX RFI and OX RFII. After 10 min of synthesis, 60% of DNA synthesized with extracts derived from pol A strains was OX RFII, which after further incubation was converted to RFI. OX RFI synthesized in vitro appears to have the same superhelical density as 4X RFI isolated in vivo. These results contrast with those obtained with t The gene A product used was purified 2000-fold by Drs. Joh-e Ikeda and A. Yudelevich of this Department. In addition, the RF replication system is capable of catalyzing net synthesis of DNA.
Proc. Natl. Acad. Sct. USA 73 (1976)
Biochemistry: Sumida-Yasumoto et al.
1891
Neutral sucrose gradient
A 2
*XRFI *XRF x
_C,2 0I
I
,
a.
'A
2 I
I?
I
x
a-
CQ Fe)
20 30 40 10 20 30 40 50 60 10 NUMBER FRACTION FIG. 4. Neutral sucrose gradient and buoyant density sedimentation analyses of BrdUrd-labeled XX DNA synthesized in vitro. DNA was synthesized with [a-32P]dATP (400 cpm/pmol), XX [3HJRFI (6 cpm/pmol), 40guM BrdUTP (in place of dTTP), and fraction II (54 lug) prepared as described in the legend of Fig. 1B. DNA was subjected to neutral sucrose gradient (A) as described in the legend of Fig. 3 and density sedimentation in neutral CsCl (B). (A) Arrows indicate the positions of reference OX [3H]RFI and [3H]RFII markers. (B) For neutral CsCl analysis, the volume was adjusted to 3 ml with 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM NaCl to which 3.7 g of CsCl was added. The mixture was sedimented for 64 hr at 35,000 rpm in a SW-50.1 rotor at 200. Fractions were collected from the bottom of the tube, and acid-insoluble material was measured. Arrows indicate the positions expected for fully heavy (H-H), half-heavy (H-L), and completely light (L-L) OX RFI DNA. vitro system in which DNA polymerase I plus DNA ligase convert M13 or OX RFII to RFI (23, 6). The latter products an in
possessed higher buoyant densities, as measured by isopycnic banding in ethidium bromide-CsCl, than the corresponding forms in vivo. We have devised complementation assays for various proteins essential in OX RFI replication. This technique, already used to isolate proteins involved in the conversion of kX174 DNA to RFII (3), has permitted us to isolate the OX gene A proteint as well as the rep give (unpublished observation). A cell-free system has been developed by modifying the procedure described in Materials and Methods in which OX RFI-dependent OX 174 DNA synthesis occurs (unpublished observation). Thus, single-stranded circular phage DNA can now be replicated through the three stages described in the introduction with cell-free preparations. We are indebted to Drs. J. Wecshler, L. Dumas, D. Denhardt, R. L. Sinsheimer, H. Hayashi, and R. Knippers for supplying E. coli strains and mutants of phage OX174. We thank Dr. S. Wickner for many suggestions. This work was supported by the National Institutes of Health, the National Science Foundation and The American Cancer Society. 1. Wickner, W. T., Brutlag, D., Schekman, R. & Kornberg, A. (1972) Proc. Natl. Acad. Sci. USA 69,965-969. 2. Wickner, R. B., Wright, M., Wickner, S. & Hurwitz, J. (1972) Proc. Natl. Acad. Sci. USA 69,3233-3237. 3. Wickner, S., Wright, M., Berkower, I. & Hurwitz, J. (1974) in DNA Replication, ed. Wickner, R. B. (Marcel Dekker, Inc., New York), pp. 195-215.
4.
Wickner, S. & Hurwitz, J. (1976) Proc. Natl. Acad. Sci. USA 73,
1053-1057. 5. Wickner, S. & Hurwitz, J. (1974) Proc. Natl. Aced. Sci. USA 71, 4120-4124. 6. Schekman, R., Weiner, A. & Kornberg, A. (1974) Science 186, 987-993. 7. Schekman, R., Weiner, H. J., Weiner, A. & Kornberg, A. (1974) 8.
9. 10. 11. 12. 13. 14.
J. Biol. Chem. 250,5859-5865. Kranias, E. G. & Dumas, L. B. (1974) J. Virol. 13, 146-154. Dumas, L. B. & Miller, C. A. (1973) J. Virol. 11, 848-855. McFadden, G. & Denhardt, D. T. (9174) J. Virol. 14,1070-1075. Truitt, C. L. & Walker, J. R. (1974) Biochem. Biophys. Res. Commun. 61,1036-1042. Taketo, A. (1975) Mol. Gen. Genet. 139,285-291. Denhardt, D. T., Dressler, D. H. & Hathaway, A. (1967) Proc. Nati. Acad. Sci. USA 57,813-820. Lindquist, B. H. & Sinsheimer, R. L. (1967) J. Mol. Biol. 30,
69-80. 15. Hurwitz, J.- & Wickner, S. (1974) Proc. Natl. Acad. Sci. USA 71, 6-10. 16. Sclair, M., Edgell, M. H. & Hutchinson, C. A. (1973) J. Virol. 11, 378-85. 17. Vapnek, D. & Rupp, W. D. (1971) J. Mol. Biol. 60,413. 18. Komano, T. & Sinsheimer, R. L. (1968) Biochim. Biophys. Acta 155,295. 19. Francke, B. & Ray, D. S. (1971) Virology 44, 168. 20. Sakakibara, Y. & Tomizawa, J. (1974) Proc. Natl. Acad. Sci. USA
71,802-806. 21. Goss, W. A., Deitz, W. H. & Cook, T. M. (1965) J. Bacteriol. 89, 1068-1074. 22. Staudenbauer, W. L. (1975) J. Mol. Biol. 96,201-205. 23. Geider, K. & Kornberg, A. (1974) J. Biol. Chem. 249,3999-4005.
