Molec. gen. Genet. 140, 39--50 (1975) © by Springer-Verlag 1975
Two Replication Initiation Sites on R-plasmid DNA J o r g e H. Crosa, L i n d a K. L u t t r o p p , F r e d Heffron, a n d S t a n l e y F a l k o w Department of microbiology, School of Medicine, University of Washington, Seattle, Washington Received June 7, 1975 Summary. Replicating DNA molecules of a deletion mutant of the conjugativc R-plasmid R 6 K are cleaved at a single site by the EcoRI restriction endonuclease. Electron microscope examination and measurements of the EcoRI treated replicative intermediate molecules indicate that replication can be initiated at two sites on the plasmid DNA molecule. The two sites are located at about 23 and 39 % of total length, respectively, from the EcoRI cleavage site. About 5 % of the replicating molecules use both replication initiation sites simultaneously.
Introduction T h e presence of a single u n i q u e cleavage site for t h e E c o R I r e s t r i c t i o n endonuclease in D N A of s i m i a n virus 40 (SV40) a n d in t h e p l a s m i d Col E1 has been used to establish an i n t e r n a l m a r k e r for t h e m a p p i n g a n d l o c a t i o n of t h e replicat i o n i n i t i a t i o n site of these D N A species ( F a r e e d et al., 1972 ; I n s e l b u r g st al., 1974 ; L o v e t t et al., 1974; T o m i z a w a et al., 1974). These d a t a i n d i c a t e d t h a t SV40 a n d Col E1 D N A i n i t i a t e d r e p l i c a t i o n from a u n i q u e origin of replication. H o w e v e r , while Col E1 e x t e n d e d r e p l i c a t i o n from t h e i n i t i a t i o n site in one direction, SV40 r e p l i c a t i o n was b i d i r e c t i o n a l . T h e R p l a s m i d R 6 K is u n u s u a l in t h a t it is conjugative, r e p l i c a t e s in Escherichia coli ceils as a m u l t i p l e c o p y pool (relaxed replication), a n d is of r e l a t i v e l y small size, 26 × 10 ~ d a l t o n s ( K o n t o m i c h a l o u et al., 1970). R 6 K has two sites suscept i b l e to cleavage b y E c o R I (Kupersztoch, et al., 1974) which complicates t h e m a p p i n g of t h e origin a n d d i r e c t i o n of r e p l i c a t i o n b y t h e m e t h o d e m p l o y e d for SV40 a n d Col E l . I n this p a p e r we r e p o r t t h e isolation of a d e l e t i o n m u t a n t of R 6 K called R S F 1040 which r e t a i n e d m a n y of t h e d i s t i n c t i v e features of t h e par e n t a l R 6 K p l a s m i d b u t now has o n l y one E c o R I cleavage site. R S F 1 0 4 0 can i n i t i a t e its r e p l i c a t i o n from either of two d i s t i n c t origins and, in some cases, e m p l o y s both r e p l i c a t i o n i n i t i a t i o n sites s i m u l t a n e o u s l y .
Materials and Methods Bacterial Strains and Plasmids. Escherichia coli K-12 W1485-1 F - thy- Nx r containing either R 6K (Sinr Apr) or RSF 1040 (Apr) was used in this study 1. The media employed for the growth of bacterial strains and the methods employed for bacterial matings are those described by Macrina et al., 1974. The method of Steers et aL (1959) was employed to determine the minimal inhibitory concentration of antibiotics. The Labelling o/ RSJF I040 Replicative Intermediates. E. coli W 1485-1 (RSF 1040) was prelabelled with 2-(C14) thymine (0.67 ~zCi/ml; 1.7 ~g/ml) for several generations in minimal media (Crosa et al., 1975) to a cell density of 2 X 10s cells/ml. The cells were harvested and washed at 25 ° C and resuspended in fresh thymine free medium at 37° C. In some experiments 3.0 x 10-a M
1 Star: Streptomycin resistance; Apr: Ampicillin resistance; Nxr: Nalidixic acid resistance; thy-: thymine requirement.
40
J.H. Crosa et al.
cyclic adenosine 3',5'-monophosphate was included at this point. After 30 minutes the cells were shifted to a 25°C waterbath for 10 min and given a pulse (varying between 10 and 40 seconds in different experiments) of (3H) thymidine (10 [zCi/ml; 0.05 [zg/ml). Incorporation was stopped by the addition of sodium azide (5 × 10-2 N, final concentration) and the cells were immediately frozen in a dry ice-ethanol bath. Isolation o/ Plasmid DNA. Purified covalently closed circular (CCC) plasmid DNA from cells in the logarithmic phase of growth was isolated by CsCl-ethidium bromide equilibrium centrifugation using methods which we have previously described in detail (Crosa et al., 1975). Cells pulsed with (all) thymidine were thawed, collected by centrifugation and resuspended to a density of 5 × 101° cells/ml. The cells were lysed by the procedures of Clewell and Helinski (1969) modified by the use of 0.05% Triton X-100 in place of Brij 58 and deoxycholate (Kupersztoch, Y. M., Helinski, D. R., 1973). The lysate was centrifuged at 46000 xg for 25 rain and the supernatant fluid was centrifuged to equilibrium in a CsCl-ethidium bromide gradient (Crosa et al., 1975). The gradient was fractionated and the fractions corresponding to replicative intermediates (distributed in the region of the gradient between the prelabelled CCC and open circular DNA) were pooled. EeoRI Treatment o/Plasmid DNA. Purified CCC plasmid DNA or pools containing plasmid replicative intermediate DNA were extracted (6 times) with isopropanol to remove ethidium bromide and dialyzed against 0.1 M Tris HC1, 0.05 M NaC1 pI-I 7.5. After dialysis, Mg C12 (0.01 M final concentration) and purified Ecol~I restriction endonuclease (the generous gift of H. W. Boyer) were added to 0.05ml of plasmid DNA. After 15 rain at 37°C the action of the enzyme was stopped by the addition of EDTA to a final concentration of 0.015 M. The effects and the efficiency of EcoRI digestion were monitored by electrophoresis on agarose gels (Greene ctal., 1974). Electron Microscopy o/Plasmid DNA. Cytochrome C was added to a final concentration of 0.1 mg/ml to either untreated or EcoRI digested DNA and the DNA spread by the method of Davis et al. (1971). The DNA was picked up on parlodian coated grids, rotary shadowed with P t : P d (80:20) and examined with a JEOL 100B electron microscope. The method of electron microscope DNA-DNA heteroduplex analysis has been described previously (Heffron et al., 1975). D N A - D N A Homology Studies. Labelled plasmid and unlabelled total bacterial DNA were prepared and the degree of nueleotide sequence homology between plasmid DNA was analyzed by the Si nuclease assay as described by Crosa et al. (1973).
Results Genetic and Molecular Properties o/ R S F I040 D u r i n g the r o u t i n e e x a m i n a t i o n of a stock culture of W1485-1 ( R 6 K ) one clone was i n c i d e n t a l l y observed to have lost its low level resistance to streptomycin, although r e t a i n i n g plasmid m e d i a t e d ampicillin resistance. Because of its p o t e n t i a l usefulness for plasmid i n c o m p a t i b i l i t y studies we e x a m i n e d i n some detail the plasmid w i t h i n this exceptional clone as s u m m a r i z e d in Table 1. This plasmid, which we now call R S F 1 0 4 0 , was f o u n d to be significantly altered in several respects from the p a r e n t a l R 6 K plasmid. As judged b y both sucrose grad i e n t analysis a n d electron microscope contour length measurements, R S F 1 0 4 0 was 17.3 × 106 daltons in size as compared to 26 × 106 dMtons for t ~ 6 K . Reciprocal D N A - D N A h y b r i d i z a t i o n studies (shown i n Table 1) as well as electron microscope hcteroduplex analysis confirmed t h a t R S F 1040 was formed b y the deletion of almost 9 × 10~ daltons of D N A from R 6 K . Thus, a heteroduplex molecule formed between R 6 K a n d R S F 1 0 4 0 shows t h a t the two plasmids are t o t a l l y homologous except for a single c o n t i n u o u s region corresponding to a p p r o x i m a t e l y 33% of t h e R 6 K genome (Fig. 1). Despite this s u b s t a n t i a l deletion of genetic
Two Replication Initiation Sites on g-plasmid DNA
41
Fig. 1. Heteroduplex between g 6 K and RSF 1040 DNA. Heteroduplex DNA molecules were obtained by mixing 0.1 to 0.2 ~zg of each X-ray nicked (Helfron et al., 1975) plasmid DNA in 0.25 ml of a solution containing 0.1 N NaOH and 0.01 M EDTA (pH 12.4) for 10 rain and then bringing down the pH to 8.5 with 2 3/[ Tris HC1 pH 7.1. The DNA was reannealed by adding 0.25 ml of formamide (Mallinckrodt) and allowing it to stand for 3 hours at 25 ° C. Spreading and shadowing were carried out as described (Heffron et al., 1975). Grids were examined with a JEOL Model 100 B electron microscope. The arrow shows a single stranded loop in the heteroduplex DNA molecule. The shared duplex region is about 70% of R 6 K DNA
m a t e r i a l R S F 1040 still m a i n t a i n e d m a n y of t h e d i s t i n c t i v e p r o p e r t i e s of R 6 K , i t was conjugative, p r e s e n t as m u l t i p l e copies w i t h i n host b a c t e r i a l cells a n d CCC molecules of R S F 1040 showed a ' r e l a x a t i o n c o m p l e x ' as e v i d e n c e d b y a t r a n s i t i o n to open circular D N A after a p p r o p r i a t e t r e a t m e n t (Table 1). R S F 1040 shows a 10-fold increase in conjugal t r a n s f e r in E. coli when comp a r e d t o R 6K. I n a d d i t i o n , t h e n u m b e r of p l a s m i d D N A molecules p e r chromosome e q u i v a l e n t is h i g h e r for R S F 1040 (Table 1). I t was suggested t h a t g 6 K conjugal t r a n s f e r is d e p e n d e n t on t h e n u m b e r of p l a s m i d copies per c h r o m o s o m e e q u i v a l e n t (Macrina et al., 1974). Our d a t a confirm a d e p e n d e n c e b e t w e e n p l a s m i d c o p y n u m b e r a n d conjugal transfer, a l t h o u g h R S F 1040 is of a smaller molecular weight t h a n t h e p a r e n t a l 1%6 K D N A . I t r e m a i n s to be seen w h e t h e r t h e increased R S F 1040 conjugal t r a n s f e r is due to some o t h e r a l t e r a t i o n in its genetic characteristics. I n a d d i t i o n to its smaller size, R S F 1040 was f o u n d to possess a tool fraction g u a n i n e + c y t o s i n e (G + C) c o n t e n t of 0.42 as c o m p a r e d to 0.45 for R 6 K . One can d i r e c t l y calculate, therefore, t h a t t h e D N A d e l e t e d from l g 6 K to form R S F 1040 was on t h e a v e r a g e 0.51 tool f r a c t i o n G ~-C. F u r t h e r m o r e , analysis of E c o R I t r e a t e d R S F 1040 D N A b y agarose gel electrophoresis r e v e a l e d t h a t it h a d b u t one site susceptible to t h e a c t i o n of t h i s r e s t r i c t i o n endonuclease. This m a y be seen in Fig. 2 which illustrates t h a t R 6 K is cleaved to two linear f r a g m e n t s of 10 × l 0 s a n d 16 × 10 s d a l t o n s c o m p a r e d to t h e single linear f r a g m e n t of R S F 1040 g e n e r a t e d b y E c o R I t r e a t m e n t . I t was t h i s l a t t e r p r o p e r t y in p a r t i c u l a r which
42
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Two Replication Initiation Sites on R-plasmid DNA J o r g e H. Crosa, L i n d a K. L u t t r o p p , F r e d Heffron, a n d S t a n l e y F a l k o w Department of microbiology, School of Medicine, University of Washington, Seattle, Washington Received June 7, 1975 Summary. Replicating DNA molecules of a deletion mutant of the conjugativc R-plasmid R 6 K are cleaved at a single site by the EcoRI restriction endonuclease. Electron microscope examination and measurements of the EcoRI treated replicative intermediate molecules indicate that replication can be initiated at two sites on the plasmid DNA molecule. The two sites are located at about 23 and 39 % of total length, respectively, from the EcoRI cleavage site. About 5 % of the replicating molecules use both replication initiation sites simultaneously.
Introduction T h e presence of a single u n i q u e cleavage site for t h e E c o R I r e s t r i c t i o n endonuclease in D N A of s i m i a n virus 40 (SV40) a n d in t h e p l a s m i d Col E1 has been used to establish an i n t e r n a l m a r k e r for t h e m a p p i n g a n d l o c a t i o n of t h e replicat i o n i n i t i a t i o n site of these D N A species ( F a r e e d et al., 1972 ; I n s e l b u r g st al., 1974 ; L o v e t t et al., 1974; T o m i z a w a et al., 1974). These d a t a i n d i c a t e d t h a t SV40 a n d Col E1 D N A i n i t i a t e d r e p l i c a t i o n from a u n i q u e origin of replication. H o w e v e r , while Col E1 e x t e n d e d r e p l i c a t i o n from t h e i n i t i a t i o n site in one direction, SV40 r e p l i c a t i o n was b i d i r e c t i o n a l . T h e R p l a s m i d R 6 K is u n u s u a l in t h a t it is conjugative, r e p l i c a t e s in Escherichia coli ceils as a m u l t i p l e c o p y pool (relaxed replication), a n d is of r e l a t i v e l y small size, 26 × 10 ~ d a l t o n s ( K o n t o m i c h a l o u et al., 1970). R 6 K has two sites suscept i b l e to cleavage b y E c o R I (Kupersztoch, et al., 1974) which complicates t h e m a p p i n g of t h e origin a n d d i r e c t i o n of r e p l i c a t i o n b y t h e m e t h o d e m p l o y e d for SV40 a n d Col E l . I n this p a p e r we r e p o r t t h e isolation of a d e l e t i o n m u t a n t of R 6 K called R S F 1040 which r e t a i n e d m a n y of t h e d i s t i n c t i v e features of t h e par e n t a l R 6 K p l a s m i d b u t now has o n l y one E c o R I cleavage site. R S F 1 0 4 0 can i n i t i a t e its r e p l i c a t i o n from either of two d i s t i n c t origins and, in some cases, e m p l o y s both r e p l i c a t i o n i n i t i a t i o n sites s i m u l t a n e o u s l y .
Materials and Methods Bacterial Strains and Plasmids. Escherichia coli K-12 W1485-1 F - thy- Nx r containing either R 6K (Sinr Apr) or RSF 1040 (Apr) was used in this study 1. The media employed for the growth of bacterial strains and the methods employed for bacterial matings are those described by Macrina et al., 1974. The method of Steers et aL (1959) was employed to determine the minimal inhibitory concentration of antibiotics. The Labelling o/ RSJF I040 Replicative Intermediates. E. coli W 1485-1 (RSF 1040) was prelabelled with 2-(C14) thymine (0.67 ~zCi/ml; 1.7 ~g/ml) for several generations in minimal media (Crosa et al., 1975) to a cell density of 2 X 10s cells/ml. The cells were harvested and washed at 25 ° C and resuspended in fresh thymine free medium at 37° C. In some experiments 3.0 x 10-a M
1 Star: Streptomycin resistance; Apr: Ampicillin resistance; Nxr: Nalidixic acid resistance; thy-: thymine requirement.
40
J.H. Crosa et al.
cyclic adenosine 3',5'-monophosphate was included at this point. After 30 minutes the cells were shifted to a 25°C waterbath for 10 min and given a pulse (varying between 10 and 40 seconds in different experiments) of (3H) thymidine (10 [zCi/ml; 0.05 [zg/ml). Incorporation was stopped by the addition of sodium azide (5 × 10-2 N, final concentration) and the cells were immediately frozen in a dry ice-ethanol bath. Isolation o/ Plasmid DNA. Purified covalently closed circular (CCC) plasmid DNA from cells in the logarithmic phase of growth was isolated by CsCl-ethidium bromide equilibrium centrifugation using methods which we have previously described in detail (Crosa et al., 1975). Cells pulsed with (all) thymidine were thawed, collected by centrifugation and resuspended to a density of 5 × 101° cells/ml. The cells were lysed by the procedures of Clewell and Helinski (1969) modified by the use of 0.05% Triton X-100 in place of Brij 58 and deoxycholate (Kupersztoch, Y. M., Helinski, D. R., 1973). The lysate was centrifuged at 46000 xg for 25 rain and the supernatant fluid was centrifuged to equilibrium in a CsCl-ethidium bromide gradient (Crosa et al., 1975). The gradient was fractionated and the fractions corresponding to replicative intermediates (distributed in the region of the gradient between the prelabelled CCC and open circular DNA) were pooled. EeoRI Treatment o/Plasmid DNA. Purified CCC plasmid DNA or pools containing plasmid replicative intermediate DNA were extracted (6 times) with isopropanol to remove ethidium bromide and dialyzed against 0.1 M Tris HC1, 0.05 M NaC1 pI-I 7.5. After dialysis, Mg C12 (0.01 M final concentration) and purified Ecol~I restriction endonuclease (the generous gift of H. W. Boyer) were added to 0.05ml of plasmid DNA. After 15 rain at 37°C the action of the enzyme was stopped by the addition of EDTA to a final concentration of 0.015 M. The effects and the efficiency of EcoRI digestion were monitored by electrophoresis on agarose gels (Greene ctal., 1974). Electron Microscopy o/Plasmid DNA. Cytochrome C was added to a final concentration of 0.1 mg/ml to either untreated or EcoRI digested DNA and the DNA spread by the method of Davis et al. (1971). The DNA was picked up on parlodian coated grids, rotary shadowed with P t : P d (80:20) and examined with a JEOL 100B electron microscope. The method of electron microscope DNA-DNA heteroduplex analysis has been described previously (Heffron et al., 1975). D N A - D N A Homology Studies. Labelled plasmid and unlabelled total bacterial DNA were prepared and the degree of nueleotide sequence homology between plasmid DNA was analyzed by the Si nuclease assay as described by Crosa et al. (1973).
Results Genetic and Molecular Properties o/ R S F I040 D u r i n g the r o u t i n e e x a m i n a t i o n of a stock culture of W1485-1 ( R 6 K ) one clone was i n c i d e n t a l l y observed to have lost its low level resistance to streptomycin, although r e t a i n i n g plasmid m e d i a t e d ampicillin resistance. Because of its p o t e n t i a l usefulness for plasmid i n c o m p a t i b i l i t y studies we e x a m i n e d i n some detail the plasmid w i t h i n this exceptional clone as s u m m a r i z e d in Table 1. This plasmid, which we now call R S F 1 0 4 0 , was f o u n d to be significantly altered in several respects from the p a r e n t a l R 6 K plasmid. As judged b y both sucrose grad i e n t analysis a n d electron microscope contour length measurements, R S F 1 0 4 0 was 17.3 × 106 daltons in size as compared to 26 × 106 dMtons for t ~ 6 K . Reciprocal D N A - D N A h y b r i d i z a t i o n studies (shown i n Table 1) as well as electron microscope hcteroduplex analysis confirmed t h a t R S F 1040 was formed b y the deletion of almost 9 × 10~ daltons of D N A from R 6 K . Thus, a heteroduplex molecule formed between R 6 K a n d R S F 1 0 4 0 shows t h a t the two plasmids are t o t a l l y homologous except for a single c o n t i n u o u s region corresponding to a p p r o x i m a t e l y 33% of t h e R 6 K genome (Fig. 1). Despite this s u b s t a n t i a l deletion of genetic
Two Replication Initiation Sites on g-plasmid DNA
41
Fig. 1. Heteroduplex between g 6 K and RSF 1040 DNA. Heteroduplex DNA molecules were obtained by mixing 0.1 to 0.2 ~zg of each X-ray nicked (Helfron et al., 1975) plasmid DNA in 0.25 ml of a solution containing 0.1 N NaOH and 0.01 M EDTA (pH 12.4) for 10 rain and then bringing down the pH to 8.5 with 2 3/[ Tris HC1 pH 7.1. The DNA was reannealed by adding 0.25 ml of formamide (Mallinckrodt) and allowing it to stand for 3 hours at 25 ° C. Spreading and shadowing were carried out as described (Heffron et al., 1975). Grids were examined with a JEOL Model 100 B electron microscope. The arrow shows a single stranded loop in the heteroduplex DNA molecule. The shared duplex region is about 70% of R 6 K DNA
m a t e r i a l R S F 1040 still m a i n t a i n e d m a n y of t h e d i s t i n c t i v e p r o p e r t i e s of R 6 K , i t was conjugative, p r e s e n t as m u l t i p l e copies w i t h i n host b a c t e r i a l cells a n d CCC molecules of R S F 1040 showed a ' r e l a x a t i o n c o m p l e x ' as e v i d e n c e d b y a t r a n s i t i o n to open circular D N A after a p p r o p r i a t e t r e a t m e n t (Table 1). R S F 1040 shows a 10-fold increase in conjugal t r a n s f e r in E. coli when comp a r e d t o R 6K. I n a d d i t i o n , t h e n u m b e r of p l a s m i d D N A molecules p e r chromosome e q u i v a l e n t is h i g h e r for R S F 1040 (Table 1). I t was suggested t h a t g 6 K conjugal t r a n s f e r is d e p e n d e n t on t h e n u m b e r of p l a s m i d copies per c h r o m o s o m e e q u i v a l e n t (Macrina et al., 1974). Our d a t a confirm a d e p e n d e n c e b e t w e e n p l a s m i d c o p y n u m b e r a n d conjugal transfer, a l t h o u g h R S F 1040 is of a smaller molecular weight t h a n t h e p a r e n t a l 1%6 K D N A . I t r e m a i n s to be seen w h e t h e r t h e increased R S F 1040 conjugal t r a n s f e r is due to some o t h e r a l t e r a t i o n in its genetic characteristics. I n a d d i t i o n to its smaller size, R S F 1040 was f o u n d to possess a tool fraction g u a n i n e + c y t o s i n e (G + C) c o n t e n t of 0.42 as c o m p a r e d to 0.45 for R 6 K . One can d i r e c t l y calculate, therefore, t h a t t h e D N A d e l e t e d from l g 6 K to form R S F 1040 was on t h e a v e r a g e 0.51 tool f r a c t i o n G ~-C. F u r t h e r m o r e , analysis of E c o R I t r e a t e d R S F 1040 D N A b y agarose gel electrophoresis r e v e a l e d t h a t it h a d b u t one site susceptible to t h e a c t i o n of t h i s r e s t r i c t i o n endonuclease. This m a y be seen in Fig. 2 which illustrates t h a t R 6 K is cleaved to two linear f r a g m e n t s of 10 × l 0 s a n d 16 × 10 s d a l t o n s c o m p a r e d to t h e single linear f r a g m e n t of R S F 1040 g e n e r a t e d b y E c o R I t r e a t m e n t . I t was t h i s l a t t e r p r o p e r t y in p a r t i c u l a r which
42
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