Molec. gen. Genet. 136, 273--289 (1975) © by Springer-Verlag 1975

Plasmid ColE1 DNA Replication in Escherichia coli Strains Temperature-sensitive

for DNA Replication

,John Collins, Peter Williams, and Donald R. Helinski

Department of Biology, University of California, San Diego, La Jolla~ California, U.S.A.

Received October 15, 1974

Summary. Mutants of the dnaA, dnaC, dnaD, polC, dnaF and dnaG gene loci were tested for their capacity for colicinogenic plasmid E1 (ColE1) replication at a non-permissive tem- perature. It was found that ColEl replication was independent of the dnaA gene function and dependent on dnaC, D, F and G. ColE1 replication in the polC mutant E486 continued for several hours but at a greatly reduced rate. No effect was found of the dnaG mutation on thymine-deprivation-induced "priming" of ColE1 replication at the non-permissive tem- perature. The mutants also were tested for aberrant replication intermediates of plasmid DNA as well as a temperature sensitive supercoiled DNA-protein relaxation complex. RNA- containing supercoils were found to accumulate in a poIC mutant also blocked for protein synthesis.

Introduction

The replication of the colicinogenic plasmid E1 (ColE1) has been shown to be dependent on chromosomal genes (Kingsbury and Helinski, 1973). Mutations in these genes result in a defect specifically in plasmid DNA synthesis. One class of these mutants was found to be temperature-sensitive for DNA polymerase I (Kingsbury and Helinski, 1973). Mutants in this class show an immediate cessation specifically in plasmid ColE1 replication at 42 °. Goebel (1970a, b, 1972, 1973) and Goebel and Schrempf (1972) have reported the effect on ColE1 DNA synthesis of some of the main groups of chromosomal mutants conditional for chromosomal DNA synthesis. In these studies it was reported that ColE1 replication is dependent on dnaA and to some extent on the dnaB and dnaC gene products while being entirely independent of the dnaE (polC) gene product, DNA polymerase I I I . The findings present here, which do not include studies of dnaB mutants, lead to some contradictory conclusions from these earlier reports and extend the study to dnaD, dnaF and dnaG mutants. In this report ColE1 replication is shown to be entirely independent of the dnaA function but dependent on the dnaC, dnaD, polC, dnaF and dnaG functions, The dependency onpolC is demonstrated as not being absolute, but in the light of data on the leakiness of the mutation in these strains (Tait and Smith, 1974) the results are taken as a positive indication of some dependency.

Materials and Methods M9 casamino acids and LB broth media used in the experiments have been described

previously, along with a procedure for preconditioning the medium (Collins and Pritchard, 1973). Lo-lo phosphate medium has been previously described (Williams et al., 1973). ~4C- thymine (2.2 ~g/ml, 47.5 mCi/mMole) was added to either minimal or LB broth media to prelabel the DNA for three or four generations prior to the temperature shift. From the

19 ~[olec. gen. Genet. 136

274 J. Collins et al.

observed specific activity of the DNA there appeared to be about 4 ~g/ml of thymine in the LB broth, all-thymine (50 e/mMole) was added one generation before the temperature shift (at a Klettsa 0 of approximately 25) to a final specific activity of 0.1 to 0.5 C/mMole. At the time of the temperature shift the cells were either diluted two-fold into media at 50 ° to give a final temperature of either 40 or 42 ° or samples were centrifuged and resuspended in LB broth (with or without preconditioning) at 42 °. At 42 ° the all-thymine specific activity was adjusted to be the same as at the permissive temperature. I t should be noted that where the cells had been centrifuged and resuspended, 14C-thymine was replaced by unlabeled thymine at the same concentration. Where a two-fold dilution was used, the 3H to laC-thymine ratio was effectively doubled at the non-permissive temperature compared to the permissive temperature. In calculating the increments in DNA at the non-permissive temperature, the relative specific activities of the isotopes were taken into consideration. In each case the all-thymine pre-shift labeling at the permissive-temperature was used to normalize sub- sequently observed aH/14C ratios. Samples were collected on Whatman No. 1 filter paper, washed in 5% trichloracetie acid, ethanol and then ether. The scintillation fluid contained 5 g of 2.5 diphenytoxazole per litre of toluene.

For the preparation of supereoiled ColE1 DNA cells from 10 to 15 ml of culture were resuspended in 1 ml of 25% sucrose 50 mM Tris (pH 8.0). After 5 rain 0.4 ml of 0.25 m EDTA was added. After a further 10 min, 1.6 ml of lytic mixture (0.2% Triton X-100; 0.0625 EDTA; 0.05 M TRIS pH 8.0) was added and lysis allowed to complete with some shaking for 10 min. Cleared lysates were prepared in 35 ml polypropylene tubes by eentrifugation at 20000 rpm in a Sorvall SS-34 rotor at 0 °. The cleared lysate (3 ml) was mixed with 3.55 g cesium chloride, 0.4 ml water and 0.5 ml ethidium bromide (3 mg/ml). The isopycnie gradient was prepared by eentrifugation at 40000 rpm in a Ti50 rotor at 15 ° for 40 hr. Gradients were collected by controlled dripping through a stainless steel needle to give 30 to 50 fractions per gradient. After removing the ethidium bromide with CsC1 saturated isopropanol, fractions pooled from these gradients were dialyzed against TES buffer (Tris 0.05 M, pH 8.0, EDTA 0.05 M and NaC1 0.1 M) for 4 hrs at 4 °. The dialyzed samples were centrifuged on 5-20% sucrose (containing TES and 0.5 M NaCl) gradients for 120 rain at 5 × 104 rpm in an SW 50.1 rotor at 15 °. In some cases cleared lysates (0.2 ml) were centrifuged directly on 20-31% sucrose (containing TES and 0.5 M NaC1) for 210 rain at 5 × 104 rpm in an SW 50.1 rotor at 15 ° .

Alkali and RNase sensitivity of supercoiled ColE1 DNA were assayed as previously described (Blair et al., 1972). The heat induced relaxation of the eomplexed supercoiled DNA was compared to differentially-labeled ColE1 relaxation complex from a wild-type strain in cleared lysate preparations. ColE1 complex from cleared lysates of wild-type and mutant strains were incubated together for 20 and 40 minutes at 50°C. The SDS relaxability sub- sequent to these heat incubations was also measured as described previously (Clewell and ttelinski, 1969). Incubation mixtures were analyzed for supereoiled and open circular molecules on 20-31% sucrose (containing TES and 0.5 M NaC1) gradients by eentrifugation for 4 hrs at 5 × 104 rpm in an SW 50.1 rotor.

Results

1. Analysis o/Colicinogenic Wild-type, dnaA and dnaC Mutant Strains

The parenta l and colicinogenic derivat ives of CR34 and the dna m u t a n t s shown in Table 1 were tested for the presence of supercoiled plasmids other t h a n ColE1. No supercoils were found by dye-cesium chloride equi l ibr ium gradient analysis of cleared lysates of the parenta l s t rains a t the level of detect ion of 0.005% of the total DNA. Supereoiled DNA, prepared by dye-cesium chloride gradient centr i fugat ion from cleared lysates of the colicinogenie strains, sedimented only as a single peak corresponding to supercoiled ColE1 DNA when r u n on

alkaline sucrose gradients. The detailed kinetics of plasmid synthesis subsequent to a tempera ture shift was

studied in these colicinogenic strains. Ini t ia l ly , experiments were carried out in

ColE1 DNA Replication in dna Mutants

Table 1. Bacterial strains tested for their effect on ColE1 replication

275

Designation dna mutation Strain Non-permissive references temperature

CR34 wild type [1, 5] - - CRT484 dnaA46 [2, 5, 9] 42 ° CRT15 dnaA83 [2, 3, 5, 9] 42 ° PC1 dnaC1 [4, 5] 40 ° CT248 dnaC248 [7] 42 ° PC7 dnaD7 [4, 5] 40 ° E486 dnaE486 [5] 42.5 ° El01 dnaFlOl [5] 42.5 ° BT308 dnaG308 [5, 6] 42 °

All strains were thymine requiring and PolA +. References refer to 1) Blair etal. (1971); 2) Hirota etal. (1968); 3) Nandadasa and Pritchard (1970); 4) Carl (1970); 5) Wechsler and Gross (1971); 6) Bonhoeffer (1966); 7) Rodriguez, etal. (1973); 8) Marinus and Adelberg (1970); 9) Kohiyama (1968).

LB broth, pre-labeled continuously with 14C-thymine. At the t ime of the tempera- ture shift the cells were washed to remove the 14C-thymine and resuspended in LB broth with 3H-thymine a t the non-permissive temperature. To facilitate the inter- pretation of the 8H/14C ratios in terms of amount of replication, 3H-thymine was added for one generation time prior to the temperature shift at the same specific act ivi ty as was used during the post-shift conditions. Exceptions to these general labeling conditions are noted with each mutan t group.

In addition to measuring the kinetics of replication, the accumulation of aberrant plasmid replication intermediates was investigated by at tempting to detect (i) supercoiled catenates tha t are characterized by a high sedimentation value in sucrose gradients, (ii) open and closed circular catenates consisting of interlocked DNA molecules tha t are found in an intermediate position between supercoiled and open circular DNA in a dye-cesium chloride gradient, (iii) RNA- containing supercoiled DNA molecules that are converted to the open-circular DNA form by alkali or RNase treatment, and (iv) temperature sensitivity relaxa- tion complex of supercoiled ColE1 DNA and protein.

CR34 Dna÷(ColE1) shows continued synthesis of plasmid and chromosomal DNA at 42 ° as the culture approaches the stat ionary phase (Fig. 1). Plasmid synthesis closely paralleled mass increase into the early stat ionary phase and then carried on for nearly one and a half rounds of replication after mass increase had stopped. In rich broth the completion of rounds of replication from a multiforked chromosome apparent ly maintains the overall plasmid to chromosome ratio into late s tat ionary phase. No accumulation of dimers or catenated DNA forms was observed. No significant alkali-sensitivity of the supercoiled DNA isolated from cultures incubated for seven hours at 42 ° was detected.

Of the initiation mutants tested ColE1 DNA synthesis was found to continue at a high rate only in the dnaA group, namely CRT484(dnaA46) and CRT83(dnaA83) (Figs. 2 and 3). ColE1 replication has been found to continue a t a high rate for at least two hours at 42 ° in CRT484 (Sparks, R., personal communication). Table 2

19"

276 J . Collins et at.

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Fig. 1. Plasmid and chromosomal DNA synthesis in CR34 (ColE1). The Cells were grown a t 42 ° in LB glucose broth: Chromosomal DNA =---, plasmid DNA , - - , , Kletta40 o--o

3.00i

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Fig. 2A and B. Plasmid and chromosomal DNA synthesis in dnaA mutants . (A) CRT83 (dnaA83) (ColE1) and (B) CRT484 (dnaA46) (ColE1) a t 42 ° in LB glucose broth. The tem- perature shift for CRT83 ColE1 was carried out by resuspending centrifuged cells a t 42 ° in LB bro th ; for CRT484 the shift was made by two-fold dilution into hot LB broth to give a

final temperature of 42 °. Chromosomal DNA , - - . , plasmid DNA ~--~, and Klet%~ o o--o

ColE1 DNA Replication in dna Mutants 277

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Fig. 3A--D. Plasmid and chromosomal DNA synthesis in dnaC and dnaD mutants. (A) CI%284 (dnaC) (ColE1) in M9 casamino acids glycerol, (B) CT284 (dnaC)(ColE1) in M9 easamino acids glucose, (C) PC1 (dnaC1)(ColE1) in M9 casamino acid glucose and (D) PC7 (dnaD) CotE1) in M9 easamino acids glucose. Temperature shifts in all cases were carried out by a

two-fold dilution, Chromosomal DNA .--,, plasmid DNA ,--~, and Klett540 o--o

shows the generation times of the initiation mutants at permissive and non- permissive temperatures, as well as the increments in chromosomal DNA observed after the temperature shift. The final column in Table 2 shows the calculated time for a single replication fork to traverse the chromosome at the permissive tem- perature assuming that all rounds of replication in progress are completed at the higher temperature. Pritchard and Zaritsky (1970) found that the time required for a replication fork to complete a round of replication is about 65 to 70 min at 37 ° in a thymine requiring strain of E. coli K12 in media with about 2 to 4 ~g/ml of thymine. Assuming that the rate of replication of DNA obeys the usual rule of doubling with each 10 ° increase in temperature, then the corresponding times to

278 J. Collins et al.

Table 2. Growth rates at permissive and non-permissive temperatures, and DNA increments at the non-permissive temperature for chromosomal initiation mutants

Mutant strain Medium Generation time Increment of Estimated DNA at non- chromosome

Permissive Non- permissive replication temper- permissive temp. time ature temperature ( % )

CR34 ColE1 LB glucose 33' 30' (42 °) 650 (Dna +)

CRT484 ColE1 LB glucose 37' 24' (42 °) 135 110' (33 °) (dnaA46)

CRT15 ColE1 LB glucose 52' 30' (42 °) 105 125' (30 °) (dnaA83)

CT248 ColE1 M9 CAA glyc 71' 81' (42 °) 39 72' (37 °) (dnaC)

CT248 ColE1 M9 CAA glu 45' 53' (42 °) 68 74' (37 °) (dnaC)

CT248 ColE1 LB glucose 31' 20' (40 °) 39 32' (32.5 °) (dnaC)

PC1 ColE1 LB glucose 96' 72' (40 °) 15 40' (30 °) ( dnaC1)

PC1 ColE1 M9 CAA glu 67' 48' (40 °) 25 45' (33 °) (dnaC1)

The estimated chromosome replication time at the permissive temperature is calculated from the observed increment in DNA at the non-permissive temperature according to the equation of Pritchard and Zaritsky (1970), assuming that the initiation of new rounds of replication are stopped immediately at the non-permissive temperature and that rounds in progress go to completion. CRT15 and CR34 were shifted to 42 ° after a centrifugation step to wash out the prelabel. All of the other initiation mutants were sensitive to such a procedure in that all DNA synthesis was shut off at the non-permissive temperature. With these other mutants, the shift to non-permissive temperature was carried out by two-fold dilution with hot medium. Except for the wild-type strain, the permissive temperature employed was as indicated in brackets in the right hand column.

complete a round of r ep l i ca t ion would be abou t 92 min a t 33 ° and 114 min a t 30 ° . The observa t ion of much longer e s t ima ted rep l ica t ion t imes would i m p l y ei ther t h a t the dna m u t a t i o n itself has some effect in lengthening the rep l ica t ion t ime a t the permissive t e m p e r a t u r e or t h a t a small a m o u n t of re in i t i a t ion is al lowed for a shor t t ime subsequent to the t e m p e r a t u r e shift . Very shor t e s t ima ted repl ica t ion t imes (less t h a n 60 min a t 33 °) i m p l y t h a t no t all the rounds of repl ica- t ion in progress a t the t ime of the t e m p e r a t u r e shif t are completed. This l a t t e r effect was observed under all condi t ions wi th PC1 (ColE1), which exhib i t s ex- t r eme ly poor growth in the r ich medium, and wi th CT248 (ColE1) in the r ich medium.

ColE1 repl ica t ion in the dnaC m u t a n t s appea red to recover as the rounds of chromosome repl ica t ion t e rmina ted , af ter a t least 40 minu tes of inh ib i t ion of synthesis . P u t a t i v e ca tena tes were observed in dye-ces ium chloride g rad ien t s a t the level of 0.03 to 0.05% of the t o t a l 14C pre- label af ter 80 minu tes a t 42 ° in g lycerol -casamino acids m e d i u m b u t in no o ther medium. The p u t a t i v e ca tena tes

ColE1 DNA Replication in dna Mutants 279

were not further investigated. Catenate and concatenate DNA forms of ColE1 have been reported to accumulate in another dnaC mutan t (PC2) but only in the presence of chloramphenieol (Goebel, 1974). All the initiation mutants shown in Table 2 with the exception of CRT83 failed to complete rounds of chromosome replication in rich medium if the cells were centrifuged to wash out the prelabel at the t ime of the temperature shift. In most cases chromosomal and plasmid DNA synthesis stopped immediately. Washing the cells at the t ime of the shift, rounds of replication were not completed in CT248 ColE1 even when the shift was carried out by two-fold dilution to a final temperature of 40 °, two degrees below the effective non-permissive temperature.

2. Analysis o/a Colicinogenic dnaD Mutant In the LB glucose medium both chromosome and ColE1 DNA replication

stopped immediately in PC7 (dnaD7) (ColE1) without any DNA breakdown or turnover. No dimers, catamers or alkali sensitive supercoils accumulated at 40 °. In another experiment carried out by a two-fold dilution in 1V[9 casamino acids glucose medium to 40 ° both chromosome and plasmid replication were shutdown for the first th i r ty minutes (Fig. 3D), but appeared to recover with similar kinetics on further incubation at the non-permissive temperature.

3. Analysis o/a Colicinogenic poIC Mutant Fig. 4 shows viability curves for E486, E486 (ColE1) and for a spontaneous

mutan t E486 Kil (ColE1) which we derived from E486 (ColE1) on the basis of its ability to survive at 42.5 °. The mass increase of E486 Kil (ColE1) ceases within th i r ty minutes at 42.5 °. Mass increase curves are shown in Fig. 5 for E486 (ColE1) and E486 Kil (ColE1). F'104 complements the region containing the dnaE486 mutat ion and gives continued replication of chromosomal DNA at 42.5 °. F ' 104 also overcomes the Kil phenotype. I t is not known if this is due to complementa- tion of the Kil mutat ion or prevention of the mass increase is only expressed by the Kil phenotype in conjunction with the dnaE mutation. ColE1 and chromo- some replication continue in the E486 ColE1 F'104 strain at the non-permissive temperature.

RNA and protein synthesis were studied in E486 ColE1 and E486 Kil (ColE1) measuring a2P-orthophosphate and 3H-leucine incorporation in Lo-lo phosphate medium at 42.5 °. I t was found tha t 3H-leucine incorporation closely followed mass increase (Klett540). As shown in Fig. 5, RNA synthesis in E486 (ColE1) lags behind protein synthesis at 42.5 ° implying a defect in RNA synthesis in addition to or consequent to the inhibition of DNA synthesis. By contrast in E486 Kil (ColE1), RNA and protein synthesis appear t ightly coupled, a situation perhaps similar to the stringent and relaxed control observed in rely- and re l - - mutants during chloramphenicol treatment. I t was concluded tha t the Kil phenotype consists of a block in protein synthesis.

Goebel (1972) has reported tha t ColE1 replication continues for several hours at 42.5 ° in the dnaElo~6 mutan t in M9 glucose casamino acids medium. As shown in Fig. 6 the initial rate of ColE1 replication in the low phosphate medium is much reduced compared to the rate of mass (Klett540) increase, although plasmid

280 J. Collins et al.

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Fig. 4. Viability curves at 42 ° for the dnaE mutants. Exponential cultures at a Klett54 o of 50 in LB glucose broth at 30 ° were centrifuged and resuspended at 42 ° in the same volume of LB broth. The abscissa shows the viable count (Nt) at any particular time divided by the initial cell viable count (No) as estimated on LB glucose plates at 33 °. dnaE4s 0 (~--o), dnaE4s 6

(ColE1) (~--,) and dnaE4s 6 KiI (ColE1) (o---o)

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Fig. 5A and B. The relative synthesis of RNA and protein in dnaE mutants. (A) dnaE4s e (ColE1) and (B) dnaE4s ~ Kil (ColE1). Cultures in Lo-lo phosphate medium were continuously labelled in the presence of 82p-orthophosphate (0.3 ~Ci/m Mole, 10 ~g/ml of orthophosphate) and 3tt-leucine. Culture volumes were three millilitres and the temperature shift was executed by transferring the culture to a prewarmed flask. Isotope incorporation was measured in 0.1 ml samples collected on Millipore filters (0.25 ~ pore size) and washed with two 10 ml aliquots of 0.1 M sodium phosphate buffer (pH 7.0). The rate of SH-leueine incorporation

followed the mass increase (Klett~40) very closely in both (A) and (B)

CotE1 DNA Replication in dna Mutants 28t

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Fig. 6 A and B. Plasmid and chromosomal DNA synthesis at 42.5 ° in dnaE mutants. (A) dnaE4s ~ (ColE1) and (B) dnaa8 s Ki l (ColE1). Chromosomal DNA synthesis in Lo-lo phosphate medium (~--.), duplicate experiments measuring ColE1 DNA synthesis in Lo-lo phosphate (~--~) and ColE1 DNA synthesis in LB glucose (~--~). The dotted line illustrates the rate of cell mass increase (Klett540) at 33 ° immediately before the temperature shift in Lo-lo phosphate medium

replication continues for several hours. In LB broth plasmid synthesis occurs a t a higher rate after an initial lag. E486 (ColE1) also showed a marked leakiness in 3H-thymine incorporation into the chromosome between one and three hours post shift, accompanied by considerable breakdown of prelabeled laC. This leakiness and prelabel breakdown was absent in the E486 Kil (ColE1) strain in which a t tempted chromosome reinitiation normally correlated with mass in- crease is presumably avoided. I t should also be noted tha t the breakdown is allowed for in calculation of the total increment in DNA, where it is assumed plasmid prelabel is not degraded. This last assumption is borne out in fact in the E486 (ColE1) strain where the yield of prelabel in the supercoiled peak remains at about 0.8-0.9% of total DNA prelabel in the absence of chromosome break- down. Fig. 7 shows the alkali-sensitivity of ColE1 DNA in the E486 Kil (ColE1) strain at various time after the shift to 42 ° . In this strain more than one-half of the plasmid daughter molecules synthesized after several hours a t 42 ° are alkali- sensitive supercoiled DNA molecules. These molecules are sensitive to a similar extent to pancreatic RNase A. The kinetics of ColE1 plasmid DNA replication in this strain are very similar to that previously reported for replication of the plasmid in the presence of chloramphenicol (Clewell, 1972). Alkali-sensitive supercoiled DNA was also detected in E486 (ColE1) but at a much smaller level. As shown in Fig. 8, the RNase sensitive nick is in the newly made daughter strand and not in the parental strand. This is consistent with previous observations, made on RNase sensitive supercoils found to accumulate in the presence of chlor- amphenicol (Williams and Helinski, manuscript in preparation). In other ex- periments it was observed tha t dimers accumulated to the extent of some 20-30% of the total supercoiled ColE1 DNA in E486 Kil (ColE1) and about one-half tha t for E486 (ColE1) at 42%

282 J. Collins et al.

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Fig. 7A and B. A]kali-sensitivity of supercoiled ColE1 DNA synthesized in dnaE mutants (A) dnaE~6 (ColE1) and (B) dnaE4s e Kil (ColE1). Cultures were labelled with x4C-thymine (0.5 ~c/ml) for five generations at 33 ° in Lo-lo phosphate medium, centrifuged and resuspended in the same volume (final Klett54 a of 70) of Lo-lo phosphate medium containing all-thymine (5 [~Ci/ml) as the sole isotopic label at 42.5 °. Samples were taken at the times indicated and cleared lysates prepared. Supercoiled ColE1 DNA was prepared as described in Materials and Methods. Supercoiled DNA was treated in the presence or absence of 0.3 M Na0H at 50 ° for twenty minutes. The conversion of the supercoiled DNA to open circular molecules, as seen on subsequent alkaline sucrose gradient centrifugation as a result of the alkali treatment, is

plotted as alkali-sensitivity. Ha-thymine (o--o) and Cl*-thymine ( . - - . ) labeled DNA

The s t r ik ing difference between the effect seen wi th these m u t a n t s and t h a t r epor ted prev ious ly is the in i t ia l r a te of p lasmid synthesis . Goebel (1972) r epor ted t h a t the in i t ia l r a te was 75 to 90% of t h a t of a D n a + s t ra in for the f irst n ine ty minutes pos t shift , whereas our results ind ica te a reduc t ion in ra te to abou t 20% of the in i t ia l r a te a t 32 ° dur ing the f i rs t hour and increas ing r ap id ly be tween one and th ree hours to a p p r o x i m a t e l y t h a t of the in i t ia l ra te . The ex ten t of ColE1 repl ica t ion af ter 7 hrs a t 42 ° lags m a r k e d l y beh ind mass increase in E486 (ColE1) bu t is fa i r ly normal for E486 Ki l (ColE 1). I n L B b ro th only abou t 20 % of the ColE1 supercoi led D N A molecules are found to be a lkal i -sensi t ive in E486 Ki l (ColE1) and only a background level of sens i t iv i ty is found for ColE1 D N A pur i f ied f rom E486 (ColE1) af ter 7 hrs a t 42 °.

4. Analys is o / a Colicinogenic dnaF Mutant

El01 (dnaF) is known to ca r ry a t empe ra tu r e sensi t ive d iphosphor ibonucleo t ide reductase and consequent ly the deoxynuc leo t ide pools are dep le ted in th is s t ra in a t 42 ° (Fuchs et al., 1972). The observed effect on bo th p la smid and chromosomal D N A synthes is in an E101 (ColE1) s t ra in was an immed ia t e reduc t ion of D N A synthesis to a l inear ra te upon shif t of the cul ture to 42 ° followed b y an exponent ia l r a te t h a t was 30% of the ra te of mass increase. On re tu rn ing to 32 °, a twelve minu te lag was seen before a new exponent ia l r a te was reached. Pulse labeled supercoi led D N A synthes ized in th is s t ra in was a lkal i r es i s tan t and no accumula-

ColE1 DNA Replication in dna Mutants 283

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20 30 40 50 60 70 80 FRACTION NUMBER

Fig. 8. Specificity of R1Nase action on the daughter strands of supercoiled CoIEI DNA in the E4s ~ Kil (ColE1) strain at 42.5 °. Cells were grown at 33 ° in Lo-lo phosphate medium con- taining MgSOa, t raM, KH2PO 4, 0.2 mM, glucose 0.4%, dephosphorylated bacto peptone, 0.4% and 2 ~zg/ml thymine. At a Klett540 of 80 the culture was diluted with an equal volume of medium at 51 °. The incubation was continued a further 4.5 hr at 42.5 ° in the presence of 14C-thymine 0.5 Ci/Mole). At this time aH-thymidine (50 Ci/mMole, 5 ~Ci/ml) was added. Fifteen minutes later the culture was frozen in the presence of 50 mM sodium azide. Super- coiled DNA molecules were prepared by dye-cesium chloride centrifugation of cleared lysates by the procedure of Blair et al. (1972) and further purified by re-running on a second dye- cesium chloride gradient. 26% of the pulse labeled supercoiled DNA molecules and 15% of the 14C-labeled supercoiled DNA molecules were converted to open circular molecules by either alkali or RNase A treatment as described by Blair et al. (1972). Purified supercoils were treated with 1 mg/ml pancreatic R•ase A at 37 ° for 60 minutes in a 0.05 M Tris buffer, pH 8.0 (0.005 M EDTA). The reaction was stopped by incubation at 37 ° with autodigested pronase at a concentration of 1 mg/ml for a further 30 minutes. The digest was layered on a 5-20% alkaline sucrose gradient containing 0.3 M NaOH and 1 M NaC1; centrifugation was for 225 minutes at 50000 rpm in a Spinco SW 50.1 rotor. About 100 fractions were collected by drops from the bottom of the centrifuge tube. The faster sedimenting peak contains unbroken single strand ColE1 circular DNA. The slower sedimenting peak contains linear (knicked

strand) ColE1 DNA (Blair et al., 1971)

t ion of dimers or catamers occurred at the non-permissive temperature . Lit t le difference was no ted between the plasmid and the chromosome in the rate of DNA synthesis a t the non-permissive tempera ture or subsequent to a period a t the non-permissive temperature .

5. Analys is o / a Colicinogenic dnaG Mutant

I n the dnaG m u t a n t s t ra in BT308 (ColE1) both chromosome and ColE1 replicat ion were blocked immedia te ly on shift ing to 42 ° wi thout high levels of DNA breakdown or tu rnover (Fig. 9). Evidence has been obta ined for a role of an R N A molecule as a pr imer of ColE1 replicat ion (Clewell et al., 1972; Blair et al., 1972). I t also has been proposed tha t the d n a G gene product is responsible for the R N A pr iming of Okazaki f ragment format ion (Lark, 1972; Olivera etal., 1973). Evidence also has been obta ined for the accumula t ion of " p r i m e d " ColE1

284 J. Collins et al.

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i

Fig. 9. Plasmid and chromosomal DNA synthesis in BT308 ColE1 (dnaG) at 42 ° in LB glucose broth. Chromosomal DNA (= =), plasmid DNA (A--A) and Klett~0 (o--o)

DNA molecules during thymine starvation (Katz and Helinski, 1974). ColE1 DNA replication was observed to undergo a nincfold stimulation immediately upon the addition of thymine after a period of thymine starvation carried out in the presence of 150 ~zg/ml of chloramphenicol (CM). The stimulation of synthesis was blocked by rifampiein (Katz and Helinski, 1974). To test whether or not the dnaG mutan t (BT308) was defective in "p r iming" ColE1 DNA synthesis, the rapidity of the shut off in replication subsequent to the temperature shift of ColE1 and chromosomal DNA replication was compared in the same experiment (Fig. 10). A test of ColE1 D N A " p r i m i n g " by this procedure depended on the demonstration tha t the dnaG mutat ion was readily reversible. Subsequent to periods a t 42 ° minus thymine with or without chloramphenicol, cultures were resuspended a t 30 ° for I ' , 3', 5', 7' or 9' before pulsing with aH-thymidine. The dnaG gene product appeared to become fully active immediately on shifting back to 30 ° .

A comparison of the stimulation of plasmid DNA replication in the dnaG and a Dna + revertant strain show that incubation at 42 ° minus thymine still allows accumulation of " p r i m e d " plasmid molecules in the dnaG mutan t (Table 3). Comparison of these cultures under conditions of pre-incubation minus thymine a t 30 ° and pulse-labeling at 42 ° incicates either tha t plasmid and chromosomal DNA replication stop within twenty to th i r ty seconds at 42 °, or are immcdiately reduced to 30 to 50?/0 of the wild-type control rate for the first minute a t 42 °. The dnaG mutat ion is, therefore, acting only at the level of propagation of the replication fork and not on the initiation of rounds of ColE1 DNA replication.

6. Test for Aberrant ColE1 DNA Forms

Analyses of cleared lysates of the mutant strains indicated tha t Catamers, concatamers or altered DNA-protein relaxation complexes were not present or

ColE1 DNA Replication in dna Mutants 285

Culture of dna_ G308(ColE1) or dn + (ColE1) in

Mg-casamino acids medium containing 4 ~g/ml thymine and 0.75

~Ci/ml of 14c-thymlne

J0° 1

Klett540 of 80

1 Pellet and resuspend cells in M9

casamlno acids medium minus

thymine__

CM (200 ~g/ml)

110' at 30 ° minus thymine

Centrifuge and resuspend at 42 ° for one minute. 3H-TdR pulse (i0 ~Cl/ml) and a 5' unlabelled TdR chase in the presence of 2 ~g/ml thymine

Fig. 10. Flow chart of the labelling regims used to test the effect of the dnaG mutation on the "pr iming" of ColE1 DNA replication during thymine starvation, and the effect of the dnaG mutatlon on rephcatmn subsequent to priming . The Dna strata was a spontaneous revertant of the dnaG308 (ColE1) strain. The "pr iming" of replication of the ColE1 DNA for replication subsequent to a period of thymine starvation occurs in the absence of protein synthesis (i.e. plus chloramphenieol--regime 3). Cells were harvested in the presence of 0.05 M sodium azide and the aH/14C ratios determined in a crude lysate (chromosomal DNA) and in the super- coiled peak after dye-cesinm chloride ultracentrifugation. The results obtained with regimes 1 and 2 are shown in Table 3. The doubling time for these strains is about 110' at 30 ° and 55' at 42 ° in this medium. The a H = T d R pulses subsequent to thymine starvation were carried out in the presence of 200 ~g/ml of deoxyguanosine and terminated by the addition of 400 ~g/ml of thymidine. The dnaG effect on DNA replication is immediately reversible on

returning to 30 ° from 42 °

CM (200 ~E/ml) No additions f

55' at 42 ° minus thymine

Ce~ nt r~fuTgdRa~dlr:s ~e~cdi~O:nf °r

10' unlabelled TdR chase in the presence of 2 ~g/ml thymine.

found to accumulate at either the permissive or non-permissive temperatures. The relaxation complex of ColE1 DNA purified from the mutan t strains was tested both for temperature and SDS detergent sensitivity (Blair et al., 1971). Incubations were carried out a t 50°C for 20' and 40' on differentially-labelled mixtures of cleared lysates f rom CR34 (ColE1) and the colieinogenie mutan t strains• Complex isolated from each mutan t s t ra in was found to have the same heat induced relaxation properties as wild type complex incubated i n the same reaction tube.

286 J. Collins et al.

Table 3. The effect of the dnaG308 mutation on the "priming" of ColE1 DNA replication during thymine starvation

Strain Thymine aH/14C Stimulation ColE1 starvation

Chromo- Super- Chromo- C o l E 1 chromosome/ somal coiled some ratio DNA ColE1 DNA

dna + 42 °, 55' 4.73 41.6 1.13 9.95 8.8 (ColE1) 42 °, 55'+CM 4.18 24.5 (1.00) 5.85 5.8

BT308 42 °, 55' 8.43 48.5 1.58 9.12 5.8 (ColE1) 42 °, 55'A-CM 5.33 45.0 (1.00) 8.43 8.4

The experimental regimes (1, 2 and 3) are shown in :Fig. 10. The relative stimulation of DNA synthesis of the chromosome and of the ColE1 plasmid was normalised with reference to the 3H/x4C ratio obtained for chromosomal DNA in regime 3. Under this regime there should be no new chromosome replication forks initiated during thymine starvation, since protein synthesis is prevented (Lark, 1972).

Discussion The results with the dnaA mutants indicate tha t the dnaA gene product is one

of the factors required for the initiation of chromosomal but not ColE1 DNA replication. This is in conflict with the previous reports which had indicated tha t ColE1 plasmid synthesis is completely dependent on the dnaA gene product in the absence of chloramphenicol. In these previous reports (Goebel, 1970, 1973a), two classes of dnaA mutan t were described with respect to replication of ColE1 DNA. The dnaA mutat ion in an E. coli 120/6 strain was found to stop ColE1 Db~A replication immediately at 42 °, while ColE1 DNA replication in the dnaA CRT46 strain shut off with similar delayed kinetics to the chromosome. The presence of the R1 drdl6 resistance transfer plasmid, or the addition of low concentrations (3 fzg/ml) of chloramphenicol, were reported to give some independence of ColE1 DNA replication from the dnaA gene product in the CRT46 strain (Goebel, 1973a). This led to the proposal tha t the dnaA gene product functioned as an antirepressor for the initiation of DNA synthesis. Replication of the R6K plasmid (Crosa, Heffron and Falkow, personal communication) and the single stranded DNA phages 6A and ~bX174 (Taketo, 1973) have also been shown to be independent of the dnaA gene product in vivo, although ~bX174 has been shown to require the dnaA gene product in vitro for complementary strand DNA synthesis on a viral single stranded template (Schekman et al., 1972). I t is not readily apparent as to the reason for the conflict between the results presented here and those previously reported by Goebel (1974). In support of the data reported in this manuscript it should be noted tha t by using double label experiments the measurements of the kinetics of plasmid DNA synthesis are free from errors which might occur due to the altered yield of supercoiled DNA known to occur at different temperatures. I t should also be noted that dnaA mutants are particularly sensitive to small differences in experimental procedure, particularly, the manner in which the temperative shifts are carried out. Special care was taken to be certain tha t chromosomal DNA synthesis followed the expected kinetics of shut off and completion of rounds.

ColE1 DNA Replication in dna Mutants 287

ColE1 DNA replication is sensitive to rifampicin (Clewell et al., 1972) and has been reported to be independent of pol I I I (dnaE, polC) (Goebel, 1972) and dependent on pol I (Kingsbury and Helinski, 1972). The dnaE4s ~ strain, however, still carries out a background level of chromosomal DNA synthesis of about 3 % the initial rate, or at 5 to 10% of the initial rate in rich broth media. The dnaE102e mutant strain used by Goebel (1972) is even more leaky and pol I I I activity can be detected in vitro at 43 ° (Tait and Smith, 1974). In view of this residual activity of polymerase I I I in these mutants it might be expected that the observed effect on DNA synthesis would be more marked on a large replicon than on a very small one, which after a short lag may compensate for under-replication by over- initiation, i.e. a low but finite rate of DNA synthesis would lead to a build up of more molecules in the process of replication. This could be highly effective in restoring a high overall rate of DNA synthesis where a relatively small plasmid such as ColE1 is present in many copies. The observation of a low rate of ColE1 DNA synthesis in the dnaE (polC) strain during the first hour at 42 °, followed by an increasing rate of synthesis subsequently, suggests that DNA polymerase I I I may in fact be required for ColE1 replication, but that high enough residual levels of the polymerase remain at 42 ° in the mutant strains employed so that small multicopied plasmids can compensate for a slow rate of chain elongation by initiating synthesis on more molecules. We suggest also that this potential for "pr iming" a large number of copies of a multicopied plasmid for replication in the absence of replication could also account for the subsequent recovery of plasmid replication in the dnaC mutant.

The a t tempt to observe accumulation of replication intermediates and aberrant molecular forms yielded only negative results. No evidence was obtained that any of the dna mutants studied code for a protein component which could alter the heat induced endonucleolytic activity associated with the ColE1 DNA-protein relaxation complex. Evidence that this complex is at least partly coded for by the plasmid itself and is indeed involved in replication of the ColE1 DNA molecule recently has been obtained from studies on ColE1 DNA replication mutants (Collins and ttelinski, manuscript in preparation). Furthermore, it has been found recently that relaxation complex is located at the origin/terminus of unidirectional ColE1 DNA synthesis (Lovett et al., 1974).

In conclusion, it was found that contrary to earlier reports the chromosomal DNA initiation gene function dnaA was not required for ColE1 DNA replication and some doubt was thrown on the independence of ColE1 replication of poly- merase III . Furthermore, ColE1 DNA replication stopped immediately on shifting to non-permissive conditions in the dnaC, dnaD and dnaG strains. Recovery with time in the dnaC strain is postulated as being due to the multicopy nature of the plasmid population accentuating particularly at later times any leakiness in the original mutation. The dnaG product was found to have no effect on the stimula- tion of plasmid DNA synthesis subsequent to thymine starvation where the thymine starvation was carried out under non-permissive conditions. The sub- strate limitation induced in the dnaF strain had no differential effect on chromo- somal and plasmid DNA synthesis. Finally, eatenates, concatenates or temperature sensitive DNA-protein relaxation complexes were not found to accumulate to any

288 J. Collins d al.

grea t ex t en t in a n y of the m u t a n t s tes ted, a l though i t should be po in ted ou t t h a t the inves t iga t ion was l imi ted to " c l ea red l y s a t e s " which do no t conta in membrane bound p lasmid DNA.

Acknowledgments. We are very grateful to Diane Etchison for her technical assistance We thank Drs. g. Wechsler, C. I. Davern, and D. W. Smith for bacterial strains. During this work John Collins was a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. This study was supported by the National Institute of Allergy and Infectious Diseases (AI-07194) and the National Science Foundation (GB-29492).

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Communica ted b y E. Bau tz

Dr. John Collins Department of Microbiology Oster Farimagsgade SA DK-2353 Copenhagen K Denmark

Dr. Peter Williams Dr. Donald R. Helinski Department of Biology University of California, San Diego La Jolla, California 92037 USA

20 Molec. gen Genet. 136