Molec. gen. Genet. 168, 27-36 (1979) © by Springer-Verlag 1979
Plasmid Replication Functions III. Origin and Direction of Replication of a "Mini" Plasmid Derived from R6-5
Richard M. Synenki*, Alfred Nordheim, and Kenneth N. Timmis Max-Planck-Institut ftir Molekulare Genetik, Ihnestrasse 63 73, D-1000 Berlin 33
Summary. Replicating D N A molecules of the mini R6-5 plasmid, pKTO71, were purified by equilibrium centrifugation in two successive ethidium bromidecaesium chloride gradients, converted to linear forms by cleavage with either HindIII or BglII restriction endonuclease, and examined in the electron microscope. Determination of the replication fork positions in 65 replicating molecules demonstrated that replication is initiated at a unique location on the plasmid and that it proceeds uni-directionally from this site. The direction of replication is such that the originproximal BglII cleavage site is replicated late or, in the case of the parent R6-5 plasmid, is such that the R-determinant region of the molecule is replicated early. The origin of replication, located by these experiments at R6-5 coordinate 98.6 kb, is clearly distinct from that of the R6-5 incompatibility determinant which has been shown to be located on an adjacent PstI-generated D N A fragment whose termini have R6-5 coordinates 96.8 and 97.9 kb. This result indicates that the incompatibility function is not an origin D N A sequence.
Introduction
The control of D N A replication is a subject that has received widespread attention but that is currently still poorly understood (for review, see Pritchard, 1978). One reason for the slow progress in this field is the difficulty of isolating, characterizing, and studying the interaction of the component elements of con* Current address: Department of Medicine, University of Chicago, 950 East 59th St., Chicago, Illinois 60637, U.S.A. For offprints contact." Dr. K.N. Timmis
trol systems. Because control of D N A replication probably occurs at the level of initiation of D N A replication, these component elements are likely to consist of a D N A sequence at which replication is initiated - an origin of replication and one or more elements that interact with this origin, either directly or via components of the replication enzyme complex. Thus far, the majority of studies on the control of initiation of D N A replication have been concerned with large replicons, such as the E. coli chromosome or the large F and R plasmids (Zeuthen and Pato, 1971; Rownd, 1969; Cooper, 1972; Davis and Helmstetter, 1973; Gustafsson and Nordstr6m, 1975) which, from the point of view of their size, are difficult to use as biochemical reagents. On the other hand, those replicons that are amenable to biochemical analysis, the genomes of small virulent bacteriophages like fd and ~b X174, are not subjected to control in the classic sense. Recently, it has been possible by means of D N A cloning methods (for review see Timmis et al., 1978c) to reduce large tightly regulated plasmid replicons to sizes that enable such replicons to be subjected to biochemical analysis (Cohen et al., 1973; Timmis et al., 1975; Lovett and Helinski, 1976). Miniplasmid replicons of this type are currently the subject of intensive investigation. In an accompanying report we describe the isolation and functional characterization of a miniplasmid from the large antibiotic resistance plasmid R6-5 (Andr6s et al., 1978). This miniplasmid contains three PstI-generated D N A fragments of R65 (Fig. 1). One fragment (PstI-1), that has a molecular length of about 9 kilobase pairs (kb), codes for resistance, to kanamycin but does not encode any essential replication functions. The other two fragments (PstI-4 and PstI-6) which have molecular lengths of 1.6 kb and 1.0 kb are both essential for autonomous replica-
0026-8925/79/0168/0027/$02.00
28
R.M. Synenki et al. R6-5 Origin of Replication. III
I
40
10 [',\
l
',
30
'
,
I
,,
§
2oy
IpKT07'I 2.5
8
12
ll6
210 2'4
2'8
32
3'6
4'0
4'4
4'8
-
I
I ".,
/
75
15
/
\
12.5
Pst I
Fig, 1. Restriction endonuclease cleavage map of the mini R6-5 plasmid pKTO71. The construction and characterization of the pKTO71 mini plasmid is described in an accompanying report (Andr6s et al., 1978)
tion and are known to be contiguous fragments in the parent R6-5 molecule. As a first step in an investigation of origin DNA sequences and their interactions with controlling elements, we have examined replicative intermediates of the pKTO71 plasmid in order to identify the location of its origin of replication. In this report we show that the origin of replication is located in the pKTO71 plasmid at a position corresponding to coordinate 98.6 kb in R6 5. Replication proceeds uni-directionally from this point towards the R-determinant region of the R6-5 molecule.
Materials and Methods Bacterial Strains and Media. The mini R6-5 plasmid pKTO71 was constructed in vitro as described previously by Andr6s et al. (1978) and introduced by transformation (Cohen et al., 1972) into strain KT450, nalidixic acid resistant derivative of Escherichia coli K-12 CR34 (Bachmann, 1972). Bacteria were grown at 37°C in M9 minimal medium (Miller, 1972) supplemented with 0.2% glucose, 0.5% casamino acids (Difco), 10 ~tg/ml vitamin B1 and 3 gg/ mI thymidine. Isolation of Replicative Intermediates. Replicating molecules of plasmid pKTO71 were prepared and isolated by a method similar to that used previously for the isolation of replicating molecules of the ColE1/pSC10I hybrid plasmid pSC134 (Cabello et al., 1976). Bacteria were grown in 500 ml cultures containing 50 gg/ml kanamycin and 10gCi of 2-14C-thymidine to an absorbance of A590=0.8, then harvested by centrifugation at 25°C and resuspended into 200 ml of medium lacking thymidine. After thymi-
i
¢. ;
..-"
/ v
10
,,
',
,,-.
2.5 4
6
8
10
i
i
12 14 76 18 20 22 24 Fraction No, Fig. 2 A and B. Isolation of plasmid pKTO71 replicative intermediates. A cleared Iysate from 3H-thymidine pulse-labeled bacteria carrying plasmid pKTO71 prepared as described in Materials and Methods was centrifuged to equilibrium in a caesium chlorideethidium bromide density gradient in a Beckman 60 Ti rotor at 40,000 rpm for 42 h at 15° C. 0.2 ml fractions were collected from the bottom of the gradient and the acid precipitable counts present in 25 ~tl aliquots determined (A). Fractions from the region of the gradient between the positions of the supercoiled and open circular non-replicating plasmid D N A (bar) were pooled and recentrifuged in a second dye caesium chloride gradient in a Beckman 50 Ti rotor at 40,000 rpm for 42 h at 15 ° C. 0.1 ml fractions were collected from the bottom of the gradient and the acid precipitable counts in 25pl'aliquots measured (B). SH and 14C CPM are indicated by dotted and continuous lines, respectively.
dine starvation by incubation at 37 ° C for 1 h, the culture was shifted to 25°C for 10min and pulsed for a period of 10 to 60s with 200gCi of 3H-thymidine. Sodium azide was added to a final concentration of 0.1 M and the culture immediately poured into a 2 litre Erlenmeyer flask that contained about 200 ml of ice and that was cooled in a dry ice-isopropanol bath. The culture was then thawed, the cells were collected by centrifugation, washed 2 times with TE buffer (10 m M Tris-HC1, pH 8.0, 1 m M EDTA) containing 10 m M N a N 3 and lysed by the procedure of Clewell and Helinski (1969) except that Triton X-100 (0.5% final concentration) replaced Brij 58-deoxycholate as the lysing detergent, and centrifugation of crude lysates was for 10 min instead of 15 min. Replicative intermediates were purified by centrifugation in two successive caesium chloride-ethidium bromide gradients (Radloff et al., 1967). Fractions of the gradient containing replicating mole-
R.M. Synenki et al. : R6-5 Origin of Replication. III
29
Fig. 3. Electron micrographs of replicating molecules of plasmid pKTO71. Molecules on the left are typical partially supercoiled structures with two open circular branches of equal length. Molecules on the right are examples of open circular replicating forms that were generated by the introduction of single strand breaks into the parental DNA strands
cules were pooled, extracted with isopropanol to remove the ethidium bromide, and dialyzed against TE buffer at 0°C for 4 h.
Restriction Endonuclease Treatment. Replicating molecules were cleaved to linear forms by treatment with 2-3 units of either HindIII or BgllI restriction endonuclease for 15 min at 25° C, The digest solutions contained 5 mM Tris-HCl, pH 7.4, and 6 mM MgClz. BglII was purchased from New England Biolabs and HindlII was purified from Haemophilus influenza strain d according to a procedure obtained from R. Roberts (personal communication). Electron Microscopy. DNA was prepared for electron microscopy by the aqueous method of Kleinschmidt (1968). Open circular pBR322 DNA molecules (2.6x 106 daltons; Bolivar et al., 1977) were added to all spreading mixtures as internal contour length standard. DNA was picked up on parlodion-coated grids, rotary shadowed with Pt:Pd (80:20) in an Edwards vacuum evaporator and examined with a Siemens 1A electron microscope. Photographs of replicating molecules were measured using a Numonix digitizer and the proportional lengths of replicated and unreplicated regions of molecules were calculated.
Results Isolation o f Replicative Intermediates F i g u r e 2 A s h o w s the d i s t r i b u t i o n o f t4C r a d i o a c t i v i t y (uniformly-labeled DNA) and 3H r a d i o a c t i v i t y ( p u l s e - l a b e l e d D N A ) o f a " c l e a r e d " b a c t e r i a l lysate c e n t r i f u g e d to e q u i l i b r i u m in a c a e s i u m c h l o r i d e - e t h i d i u m b r o m i d e d e n s i t y g r a d i e n t . A s we a n d o t h e r s have shown previously for plasmids and other circular D N A s ( S e b r i n g et al., 1971 ; R o m a n et al., 1974; Inselberg, 1974; L o v e t t et al., 1974; T o m i z a w a et al., 1974; C a b e l l o et al., 1976; C r o s a et al., 1976; T i m m i s et al., 1976; K a t z et al., 1977), a s i g n i f i c a n t p o r t i o n o f the p u l s e - l a b e l e d D N A is f o u n d in a r e g i o n o f the g r a d i e n t b e t w e e n s u p e r c o i l e d a n d o p e n c i r c u l a r non-replicating plasmid DNA. This demonstrates
30
R.M. Synenki et al. : R6-5 Origin of Replication. III
0
1 ,
2 ,
3
4 l
5 r --
6 i
7
r
8 i
9 xlO 6 Doitons i
i i
I I I g r
i
I I
I I I I t
I I 4
J
Fig. 4. Replicating molecules of plasmid pKTO71 digested with HindIII restriction endonuclease. On the left are electron micrographs of four selected molecules showing different degrees of replication. On the right is a diagramatic display of all replicating molecules that were photographed
that replicating molecules exhibit a limited affinity for ethidium bromide and therefore have closed circular parental duplex strands, i.e. are Cairns or theta forms (Cairns, 1963). To further purify the replicative intermediates, the fractions from this intermediate density region were pooled and recentrifuged in a second CsCl-ethidium bromide gradient (Fig. 2B), Examination by electron microscopy of material from the region between the supercoiled and open circular D N A from the second gradient showed replicating D N A molecules as partially supercoiled structures with two open circular regions of equal length (Fig. 3). A number of nicked circular replicating molecules were also observed and all contained a single replication " b u b b l e " or " e y e " which indicates the presence of a single origin of replication on the pKTO71 plasmid (Fig. 3).
Location of the Origin of Replication of pKT071 Relative to its' Unique HindIII and BgIII Cleavage sites The restriction endonucleases HindIII and BglII cleave the pKTO71 plasmid at unique sites whose
locations have been determined (Andr6s et al., 1978). Linear molecules composed of a symmetrical loop or " e y e " region of replicated D N A and two arms of unreplicated D N A were observed by electron microscopy after cleavage of replicating pKTO71 molecules with one of these endonucleases. Analysis of HindIII-cleaved molecules revealed that the origin of replication is approximately 45% of the length of the molecule from its unique HindIII endonuclease cleavage site and that replication proceeds uni-directionally along the longer arm (Figs. 4 and 6). Analysis of BglII-cleaved intermediates revealed that the origin of replication is located 14% of the molecule length from the unique Bg/II-cleavage site and that replication proceeds uni-directionally away from the BglII site along the longer arm (Figs. 5 and 6).
Location of the Origin of Replication in pKT071 and R6-5 Measurements of 65 linear replicative intermediates and the same number of non-replicating relaxed circular plasmid molecules demonstrated that the two types of molecules have the same contour length
R.M. Synenki et al. : R6-5 Origin of Replication. III
31
i
2
3
/..
5
6
7
l
~
i
i
i
i
9 x105Daltons
'i 4
I I I
III I III
Fig. 5. Replicating molecules of pKTO71 digested with BglII
which was measured as 11.8 kb. The origin of replication is therefore 14% of this, or 1.6 kb, from the BglII cleavage site on the pKTO71 plasmid (Fig. 7). This BglII site has previously been shown to be located 3,8kb from the ISl-proximal EcoRI site of R6-5 EcoRI fragment 2 which has been given the coordinate 100.8 kb by Timmis et al., 1978b, and hence has the R6-5 coordinate of 97.0 kb. The origin of replication is therefore located 2.2 kb from the ISlb-proxireal EcoRI site of R6-5 EcoRI fragment 2, and hence has the R6-5 coordinate of 98.6kb (Fig. 7). The orientations of PstI-generated D N A fragments 4 and 6 in the pKTO71 plasmid have been shown by restriction endonuclease cleavage analysis to be identical to those of the homologous fragments in the R6-5 plasmid. Sequence continuity and direction in the origin region of pKTO71, which was formed from separate PstI fragments of R6-5, are
therefore preserved and are identical to those in the origin region of R6-5. The direction of replication in the mini R6-5 plasmid pKTOT1 is therefore the same as that in the parent R6-5 molecule. In both plasmids, replication begins in the 1.6 kb PstI fragment and proceeds unidirectionally away from the BgIII cleavage site located in the adjacent 1.0 kb PstI fragment, i.e. R6-5 replication proceeds towards ISlb and the R-determinant region (Fig. 7).
R6-5 but Not Its Miniplasmid pKT071 Exists as a Relaxation Complex Many plasmids can be isolated from gently lysed bacteria as supercoiled DNA-protein "relaxation complexes" that undergo conversion (relaxation) to an
I
I
I
I
rr 80 ,< D ILl I-< 0__ J
~ 6o z I.L o T k-
"~ ° o -o 2°-
: o i '~- HindIE
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I---
~
AA
Plasmid Replication Functions III. Origin and Direction of Replication of a "Mini" Plasmid Derived from R6-5
Richard M. Synenki*, Alfred Nordheim, and Kenneth N. Timmis Max-Planck-Institut ftir Molekulare Genetik, Ihnestrasse 63 73, D-1000 Berlin 33
Summary. Replicating D N A molecules of the mini R6-5 plasmid, pKTO71, were purified by equilibrium centrifugation in two successive ethidium bromidecaesium chloride gradients, converted to linear forms by cleavage with either HindIII or BglII restriction endonuclease, and examined in the electron microscope. Determination of the replication fork positions in 65 replicating molecules demonstrated that replication is initiated at a unique location on the plasmid and that it proceeds uni-directionally from this site. The direction of replication is such that the originproximal BglII cleavage site is replicated late or, in the case of the parent R6-5 plasmid, is such that the R-determinant region of the molecule is replicated early. The origin of replication, located by these experiments at R6-5 coordinate 98.6 kb, is clearly distinct from that of the R6-5 incompatibility determinant which has been shown to be located on an adjacent PstI-generated D N A fragment whose termini have R6-5 coordinates 96.8 and 97.9 kb. This result indicates that the incompatibility function is not an origin D N A sequence.
Introduction
The control of D N A replication is a subject that has received widespread attention but that is currently still poorly understood (for review, see Pritchard, 1978). One reason for the slow progress in this field is the difficulty of isolating, characterizing, and studying the interaction of the component elements of con* Current address: Department of Medicine, University of Chicago, 950 East 59th St., Chicago, Illinois 60637, U.S.A. For offprints contact." Dr. K.N. Timmis
trol systems. Because control of D N A replication probably occurs at the level of initiation of D N A replication, these component elements are likely to consist of a D N A sequence at which replication is initiated - an origin of replication and one or more elements that interact with this origin, either directly or via components of the replication enzyme complex. Thus far, the majority of studies on the control of initiation of D N A replication have been concerned with large replicons, such as the E. coli chromosome or the large F and R plasmids (Zeuthen and Pato, 1971; Rownd, 1969; Cooper, 1972; Davis and Helmstetter, 1973; Gustafsson and Nordstr6m, 1975) which, from the point of view of their size, are difficult to use as biochemical reagents. On the other hand, those replicons that are amenable to biochemical analysis, the genomes of small virulent bacteriophages like fd and ~b X174, are not subjected to control in the classic sense. Recently, it has been possible by means of D N A cloning methods (for review see Timmis et al., 1978c) to reduce large tightly regulated plasmid replicons to sizes that enable such replicons to be subjected to biochemical analysis (Cohen et al., 1973; Timmis et al., 1975; Lovett and Helinski, 1976). Miniplasmid replicons of this type are currently the subject of intensive investigation. In an accompanying report we describe the isolation and functional characterization of a miniplasmid from the large antibiotic resistance plasmid R6-5 (Andr6s et al., 1978). This miniplasmid contains three PstI-generated D N A fragments of R65 (Fig. 1). One fragment (PstI-1), that has a molecular length of about 9 kilobase pairs (kb), codes for resistance, to kanamycin but does not encode any essential replication functions. The other two fragments (PstI-4 and PstI-6) which have molecular lengths of 1.6 kb and 1.0 kb are both essential for autonomous replica-
0026-8925/79/0168/0027/$02.00
28
R.M. Synenki et al. R6-5 Origin of Replication. III
I
40
10 [',\
l
',
30
'
,
I
,,
§
2oy
IpKT07'I 2.5
8
12
ll6
210 2'4
2'8
32
3'6
4'0
4'4
4'8
-
I
I ".,
/
75
15
/
\
12.5
Pst I
Fig, 1. Restriction endonuclease cleavage map of the mini R6-5 plasmid pKTO71. The construction and characterization of the pKTO71 mini plasmid is described in an accompanying report (Andr6s et al., 1978)
tion and are known to be contiguous fragments in the parent R6-5 molecule. As a first step in an investigation of origin DNA sequences and their interactions with controlling elements, we have examined replicative intermediates of the pKTO71 plasmid in order to identify the location of its origin of replication. In this report we show that the origin of replication is located in the pKTO71 plasmid at a position corresponding to coordinate 98.6 kb in R6 5. Replication proceeds uni-directionally from this point towards the R-determinant region of the R6-5 molecule.
Materials and Methods Bacterial Strains and Media. The mini R6-5 plasmid pKTO71 was constructed in vitro as described previously by Andr6s et al. (1978) and introduced by transformation (Cohen et al., 1972) into strain KT450, nalidixic acid resistant derivative of Escherichia coli K-12 CR34 (Bachmann, 1972). Bacteria were grown at 37°C in M9 minimal medium (Miller, 1972) supplemented with 0.2% glucose, 0.5% casamino acids (Difco), 10 ~tg/ml vitamin B1 and 3 gg/ mI thymidine. Isolation of Replicative Intermediates. Replicating molecules of plasmid pKTO71 were prepared and isolated by a method similar to that used previously for the isolation of replicating molecules of the ColE1/pSC10I hybrid plasmid pSC134 (Cabello et al., 1976). Bacteria were grown in 500 ml cultures containing 50 gg/ml kanamycin and 10gCi of 2-14C-thymidine to an absorbance of A590=0.8, then harvested by centrifugation at 25°C and resuspended into 200 ml of medium lacking thymidine. After thymi-
i
¢. ;
..-"
/ v
10
,,
',
,,-.
2.5 4
6
8
10
i
i
12 14 76 18 20 22 24 Fraction No, Fig. 2 A and B. Isolation of plasmid pKTO71 replicative intermediates. A cleared Iysate from 3H-thymidine pulse-labeled bacteria carrying plasmid pKTO71 prepared as described in Materials and Methods was centrifuged to equilibrium in a caesium chlorideethidium bromide density gradient in a Beckman 60 Ti rotor at 40,000 rpm for 42 h at 15° C. 0.2 ml fractions were collected from the bottom of the gradient and the acid precipitable counts present in 25 ~tl aliquots determined (A). Fractions from the region of the gradient between the positions of the supercoiled and open circular non-replicating plasmid D N A (bar) were pooled and recentrifuged in a second dye caesium chloride gradient in a Beckman 50 Ti rotor at 40,000 rpm for 42 h at 15 ° C. 0.1 ml fractions were collected from the bottom of the gradient and the acid precipitable counts in 25pl'aliquots measured (B). SH and 14C CPM are indicated by dotted and continuous lines, respectively.
dine starvation by incubation at 37 ° C for 1 h, the culture was shifted to 25°C for 10min and pulsed for a period of 10 to 60s with 200gCi of 3H-thymidine. Sodium azide was added to a final concentration of 0.1 M and the culture immediately poured into a 2 litre Erlenmeyer flask that contained about 200 ml of ice and that was cooled in a dry ice-isopropanol bath. The culture was then thawed, the cells were collected by centrifugation, washed 2 times with TE buffer (10 m M Tris-HC1, pH 8.0, 1 m M EDTA) containing 10 m M N a N 3 and lysed by the procedure of Clewell and Helinski (1969) except that Triton X-100 (0.5% final concentration) replaced Brij 58-deoxycholate as the lysing detergent, and centrifugation of crude lysates was for 10 min instead of 15 min. Replicative intermediates were purified by centrifugation in two successive caesium chloride-ethidium bromide gradients (Radloff et al., 1967). Fractions of the gradient containing replicating mole-
R.M. Synenki et al. : R6-5 Origin of Replication. III
29
Fig. 3. Electron micrographs of replicating molecules of plasmid pKTO71. Molecules on the left are typical partially supercoiled structures with two open circular branches of equal length. Molecules on the right are examples of open circular replicating forms that were generated by the introduction of single strand breaks into the parental DNA strands
cules were pooled, extracted with isopropanol to remove the ethidium bromide, and dialyzed against TE buffer at 0°C for 4 h.
Restriction Endonuclease Treatment. Replicating molecules were cleaved to linear forms by treatment with 2-3 units of either HindIII or BgllI restriction endonuclease for 15 min at 25° C, The digest solutions contained 5 mM Tris-HCl, pH 7.4, and 6 mM MgClz. BglII was purchased from New England Biolabs and HindlII was purified from Haemophilus influenza strain d according to a procedure obtained from R. Roberts (personal communication). Electron Microscopy. DNA was prepared for electron microscopy by the aqueous method of Kleinschmidt (1968). Open circular pBR322 DNA molecules (2.6x 106 daltons; Bolivar et al., 1977) were added to all spreading mixtures as internal contour length standard. DNA was picked up on parlodion-coated grids, rotary shadowed with Pt:Pd (80:20) in an Edwards vacuum evaporator and examined with a Siemens 1A electron microscope. Photographs of replicating molecules were measured using a Numonix digitizer and the proportional lengths of replicated and unreplicated regions of molecules were calculated.
Results Isolation o f Replicative Intermediates F i g u r e 2 A s h o w s the d i s t r i b u t i o n o f t4C r a d i o a c t i v i t y (uniformly-labeled DNA) and 3H r a d i o a c t i v i t y ( p u l s e - l a b e l e d D N A ) o f a " c l e a r e d " b a c t e r i a l lysate c e n t r i f u g e d to e q u i l i b r i u m in a c a e s i u m c h l o r i d e - e t h i d i u m b r o m i d e d e n s i t y g r a d i e n t . A s we a n d o t h e r s have shown previously for plasmids and other circular D N A s ( S e b r i n g et al., 1971 ; R o m a n et al., 1974; Inselberg, 1974; L o v e t t et al., 1974; T o m i z a w a et al., 1974; C a b e l l o et al., 1976; C r o s a et al., 1976; T i m m i s et al., 1976; K a t z et al., 1977), a s i g n i f i c a n t p o r t i o n o f the p u l s e - l a b e l e d D N A is f o u n d in a r e g i o n o f the g r a d i e n t b e t w e e n s u p e r c o i l e d a n d o p e n c i r c u l a r non-replicating plasmid DNA. This demonstrates
30
R.M. Synenki et al. : R6-5 Origin of Replication. III
0
1 ,
2 ,
3
4 l
5 r --
6 i
7
r
8 i
9 xlO 6 Doitons i
i i
I I I g r
i
I I
I I I I t
I I 4
J
Fig. 4. Replicating molecules of plasmid pKTO71 digested with HindIII restriction endonuclease. On the left are electron micrographs of four selected molecules showing different degrees of replication. On the right is a diagramatic display of all replicating molecules that were photographed
that replicating molecules exhibit a limited affinity for ethidium bromide and therefore have closed circular parental duplex strands, i.e. are Cairns or theta forms (Cairns, 1963). To further purify the replicative intermediates, the fractions from this intermediate density region were pooled and recentrifuged in a second CsCl-ethidium bromide gradient (Fig. 2B), Examination by electron microscopy of material from the region between the supercoiled and open circular D N A from the second gradient showed replicating D N A molecules as partially supercoiled structures with two open circular regions of equal length (Fig. 3). A number of nicked circular replicating molecules were also observed and all contained a single replication " b u b b l e " or " e y e " which indicates the presence of a single origin of replication on the pKTO71 plasmid (Fig. 3).
Location of the Origin of Replication of pKT071 Relative to its' Unique HindIII and BgIII Cleavage sites The restriction endonucleases HindIII and BglII cleave the pKTO71 plasmid at unique sites whose
locations have been determined (Andr6s et al., 1978). Linear molecules composed of a symmetrical loop or " e y e " region of replicated D N A and two arms of unreplicated D N A were observed by electron microscopy after cleavage of replicating pKTO71 molecules with one of these endonucleases. Analysis of HindIII-cleaved molecules revealed that the origin of replication is approximately 45% of the length of the molecule from its unique HindIII endonuclease cleavage site and that replication proceeds uni-directionally along the longer arm (Figs. 4 and 6). Analysis of BglII-cleaved intermediates revealed that the origin of replication is located 14% of the molecule length from the unique Bg/II-cleavage site and that replication proceeds uni-directionally away from the BglII site along the longer arm (Figs. 5 and 6).
Location of the Origin of Replication in pKT071 and R6-5 Measurements of 65 linear replicative intermediates and the same number of non-replicating relaxed circular plasmid molecules demonstrated that the two types of molecules have the same contour length
R.M. Synenki et al. : R6-5 Origin of Replication. III
31
i
2
3
/..
5
6
7
l
~
i
i
i
i
9 x105Daltons
'i 4
I I I
III I III
Fig. 5. Replicating molecules of pKTO71 digested with BglII
which was measured as 11.8 kb. The origin of replication is therefore 14% of this, or 1.6 kb, from the BglII cleavage site on the pKTO71 plasmid (Fig. 7). This BglII site has previously been shown to be located 3,8kb from the ISl-proximal EcoRI site of R6-5 EcoRI fragment 2 which has been given the coordinate 100.8 kb by Timmis et al., 1978b, and hence has the R6-5 coordinate of 97.0 kb. The origin of replication is therefore located 2.2 kb from the ISlb-proxireal EcoRI site of R6-5 EcoRI fragment 2, and hence has the R6-5 coordinate of 98.6kb (Fig. 7). The orientations of PstI-generated D N A fragments 4 and 6 in the pKTO71 plasmid have been shown by restriction endonuclease cleavage analysis to be identical to those of the homologous fragments in the R6-5 plasmid. Sequence continuity and direction in the origin region of pKTO71, which was formed from separate PstI fragments of R6-5, are
therefore preserved and are identical to those in the origin region of R6-5. The direction of replication in the mini R6-5 plasmid pKTOT1 is therefore the same as that in the parent R6-5 molecule. In both plasmids, replication begins in the 1.6 kb PstI fragment and proceeds unidirectionally away from the BgIII cleavage site located in the adjacent 1.0 kb PstI fragment, i.e. R6-5 replication proceeds towards ISlb and the R-determinant region (Fig. 7).
R6-5 but Not Its Miniplasmid pKT071 Exists as a Relaxation Complex Many plasmids can be isolated from gently lysed bacteria as supercoiled DNA-protein "relaxation complexes" that undergo conversion (relaxation) to an
I
I
I
I
rr 80 ,< D ILl I-< 0__ J
~ 6o z I.L o T k-
"~ ° o -o 2°-
: o i '~- HindIE
,z, 4o ._J t.Ll o
I---
~
AA
