Molec. gen. Genet. 142, 137--153 (1975) © by Springer-Verlag 1975

The Starting Point and Direction of Rolling-circle Replicative Intermediates of Coliphage Z DNA Seishi T a k a h a s h i Department of Biochemistry, McGill University, Montreal, Canada, tt3C 1Y6 Received June 1, 1975

Summary. Intermediates of )~ DNA replication iu the second half of the latent period after phage 2 infection were isolated and investigated in the electron microscope by denaturation mapping. The isolated replieative froms (I~F) are predominantly single branched circular DNA. The starting points of replication in these lariat molecules located at the same region as in the first round Z DNA replication. About 60% of the R F replicate from left to right and the other 40% replicate in the reverse direction. The free ends of the tails are located at many sites on the Z genome. Replicating circles with a linear DNA tail longer than one unit length of 2 genome represent about 30% of the replicating molecules. These long linear tails (concatemers) produced by the rolling-circle (Gilbert and Dressier, 1968; Eisen et al., 1968; Skalka et al., 1972; Takahashi, 1974) are one of the best candidates for a precursor DNA of progeny phage. Introduction The finding of t h e f a s t - s e d i m e n t i n g 2 D N A in a n alkaline sucrose g r a d i e n t ( S m i t h a n d S k a l k a , 1966; Y o u n g a n d Sinsheimer, 1968; Carter et al., 1969; MeClure a n d Gold, 1973) has suggested t h a t t h e r e p l i e a t i v e form (RF) f o u n d in t h e second half of t h e l a t e n t p e r i o d should be different from t h a t f o u n d in t h e first r o u n d of replication. This f a s t - s e d i m e n t i n g D N A which is longer t h a n t h e m o n o m e r l e n g t h (the D N A l e n g t h f o u n d in p h a g e particles) was also f o u n d in r e c o m b i n a t i o n deficient s y s t e m s (Skalka, 1971; E n q u i s t a n d Skalka, 1973; T a k a h a s h i , in p r e p a r a t i o n ) , s t r e n g t h e n i n g t h e a r g u m e n t t h a t this s t r u c t u r e can be p r o d u c e d b y rolling-circle r e p l i c a t i o n in t h e absence of r e c o m b i n a t i o n . Sens i t i v i t y to p h y s i c a l shearing a n d a b i l i t y to b i n d to b e n z o y l a t e d D E A E cellulose (consistent w i t h t h e existence of s i n g l e - s t r a n d e d regions) h a v e been r e p o r t e d for t h e rolling-circle s t r u c t u r e (Kiger a n d Sinsheimer, 1969). The recBC-nuelease s e n s i t i v i t y of this r e p l i c a t i v e i n t e r m e d i a t e , which is n o r m a l l y p r o t e c t e d b y t h e gain gene p r o d u c t , suggests t h a t t h e R F contains linear free ends or e x t e n d e d s i n g l e - s t r a n d e d regions ( E n q u i s t a n d Skalka, 1973; U n g e r a n d Clark, 1973). T h e n o n e x p o n e n t i a l increase of t h e r e v e r t a n t s of a ;t m u t a n t as a f u n c t i o n of t i m e also s u p p o r t t h e existence of rolling-circle R F which p r o d u c e a precoursor D N A of m a t u r e p h a g e (Eisen et al., 1968). R e c e n t l y , these rolling-circle molecules h a v e been i d e n t i f i e d a m o n g i n t r a cellular +~ D N A a t l a t e stage of 2 l a t e n t p e r i o d in b o t h infected a n d i n d u c e d cells (Takahashi, 1974; Bastia, personal communication; T a k a h a s h i , in preparation).

138

S. Takahashi

I n t h e p r e s e n t s t u d y , t h e s t a r t i n g p o i n t of r e p l i c a t i o n , e n d s of tails, a n d d i r e c t i o n of t h e rolling-circle r e p l i c a t i o n h a v e b e e n d e f i n e d , a n d a possible m o d e l for t h e origin of ~ rolling-circle is p r o p o s e d .

Materials and Methods

a) Phage and Bacterial Strains. Various virulent or clear mutants of ~ were used for the present experiments. ~vir, ,~cI90, ~b221cI26, ~clI68clII67, ~biollcI and ~virOts28 were labelled with [aI-I-methylJ-thymidine by infection of E. coli CR34 (thy-). Radioactive phage were purified by a two step centrifugation (low and high speed) followed by CsC1 density gradient centrifugation and then finally dialyzed against 0.01 M-MgSOa in D20 (ptI 7.0). The purified phage had a titer of 2 × 101~ plaque forming units/ml, and gave 2.3 × 10 7 CPM/ml. E. coli K12 derivative W3102 (gal-, su-), CR34 (thy-) or E835 (rec A56 B21 sup-) are used as hosts for the isolation of intracellular replicating ~t DNA. b) Extraction o/Replicating ~ DNA. An E. coli preeulture was grown in 5 ml of D-medium (Ogawa et al., 1968) at 37°C (or 32°C for ts mutant). D-medium contained 0.02 M-potassium phosphate buffer (pH 7.0), 0.01 ~-MgSOa, 1.6 IxM-FeSOa, 0.5 g (15NH¢)2S0~ (99 atom %; Merck, Sharp and Dohme, Montreal), 0.4 g deuterated algal whole hydrolysate in D20 (minimum 98 atom % ; Merck, Sharp and Dohme, Montreal), and 2.5 g maltose per liter of D20 (99.88 ml %; Bio-Rad, California). 0.5 ml of the preculture was inoculated into 20 ml of fresh D-medium and the cells allowed to grow to A590=0.70 (cell density; 2 × 10S/ml). The cells were centrifuged, a n d resuspended in 1 ml of Tris-MgSOa (0.01 M each) in D20. After starving cells for 30 min at 4°C, [3Hi-labelled ~ phage (in D20 ) was infected at a multiplicity of infection (MOI) of 10, and incubated at 4 ° C for 30 min for phage absorption (D20 freezes at 3.8°C). The infected cells were then diluted with 20 ml of prewarmed D-medium (32°C or 37°C), and incubated at the desired temperature (Temperature sensitive mutants were incubated at 32°C). DNA replication was terminated by pouring the cells into 20 ml of an ice cold solution containing 0.01 M-KCN, 0.15 M-NaC1, 0.015 M-sodium citrate and 10% of pyridine. Cells were then centrifuged and resuspended in 1.5 ml of a solution containing 0.15 M-MaC1, 0.015 M-sodium citrate, 0.01 M-EDTA and 2 mg of lysozyme. After freezing and thawing the suspension three times, 10 mg of Sarkosyl NL-30 (Geigy Chemical Co., N.Y.) was added; this solution was kept at 37°C for 20 rain. The lysate was finally treated with 4 mg of self digested pronase at 37 ° C for 3-5 hours. The solution was fraetionated by CsC1 density gradient centrifugation (Ti-60 rotor, Beckman; 30,000 ray/rain 3 days at 7°C) into 30 fractions, collected gently (to avoid shearing the DNA molecules) from the top using an ISCO Model 640 fraetiouator (Instrumentation Specialties Co., Nebraska). Fractions containing acid-precipitable [3H] activity were run in a second CsC1 density gradient centrefugation. The fractions containing DNA of density between full-light and half-heavy (defined as first round replication) or between half-heavy and full-heavy position (defined as second round replication) were dialyzed against 0.02 M-NaC1, 0.005 M-EDTA for 12 hours at 4°C and used for the preparation of electron microscopic specimens. c) Partial Denaturation o/Fractionated ~ DNA. Dialyzed )I DNA was partially denatured either by high pH (SchuSs and Inman, 1970) or high temperature (Inman, 1966). High pH denaturation; A buffer containing 67.8 mM-Na~C 03, 10.7 mN-EDTA, and 33.9 % formaldehyde was adjusted p H 11.0~11.5 with 5 M-NaOH. This solution was then mixed with dialyzed DNA solution and denatured at 23°C for 10 min. Thermal denaturation; Neutralized buffer containing 0.1 M-NaC1, 67.8 mM-KH2PO 4, 3.4 mM-EDTA, 10% HCHO (pH 6.73) and DNA (A269 nm=0.01) was heated at 52°C for 30 rain. Denatured solution was then quickly cooled in an ice bath and mixed with an equal volume of cold (i ~ 3 ° C) formamide and cytochrome-C (final concentration 0.01%). After warming up the mixture at room temperature ( 2 0 ~ 2 3 ° C) for 10 min, 5 microliters of the mixture was spread on a 1.1 ml drop of double-distilled water placed on a Teflon block. The spread sample was then picked up by touching a carbon coated mica disk to the surface of the drop, dipped in absolute ethanol, under warm N 2 gas dried, and finally rotary shadowed with platinum evaporated from a filament.

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Fig, 1A C. Examples of denaturation maps and weight average histograms. (A) Linear DNA derived from mature phage and their histogram (a) ; The number of molecules analyzed was 50. (B) Denaturation maps of circular/t DNA extracted from an infected cell ~nd their weight average histogram (b) ; Forty-seven circular molecules were analyzed. (C) Denaturation map of the single-branched sigma (a)-type circular 2 DNA derived from an infected cell and histogram (c); Circular DNA of 53 I~F were used for this histogram. Molecules belonging to (A) and (B) are denatured by high pH ( p H ~ l l . 0 ~ l l . 5 ) , and replicating molecules (0) are heat dengtured (52°C for 30 rain)

140

S. Takahashi

d) Electron Microscopy Measurement and Computation. Platinum-shadowed samples were examined in a Phillips-300 electron microscope at magnifications between, 5,000 and 10,000. Molecules were photographed on 35 or 70 mm film and measured; this data was used to compute denaturation maps and histograms (Schn6s and Inman, 1970). e) Identification o/the Computed Molecules. Linear molecules derived from mature phage, circular molecules and replicating circular ~ DNAs in an infected cells were compared by their lengths and denaturation patterns. About 50 linear 2 DNAs were examined; and they exhibited lengths of between 17.0--~17.5 micrometers. Their denaturation patterns showed an broad and highly frequent AT-rich segment at the ceater of the molecule and two other characteristic sites in one half of the genome. We define the region of the molecule that conrains these two sites as the right half of the molecule (SchnSs and Inman, 1970; Takahashi, 1974). Simple circular ~ DNAs and replicating circular/% DNAs also exhibited exactly same length and denaturation patterns as shown in Fig. 1. Results

a) Transition o/~ D N A Replicative Form (RF) Previous experiments have shown that it was possible to isolate replicating DNA molecules when [a2p]_ or [3H]-labelled light 2 phage infects E. coli growing in a heavy medium containing D20 and (15NH4)2S04. Under these conditions, the density of the labelled 2 DNA increases as replication progresses. These molecules were found in fractions located between light and hybrid density in a CsC1 density gradient; they exhibited a characteristic replicating structure (Ogawa et al., 1968; Sehn6s and Inman, 1970; Takahashi, unpublished). These workers found double branched theta (0)-type replicating molecules similar to those found in E. coli DNA replication (Cairns, 1963). This type of R F is believed to represent the first round of 2 DNA replication, which generates a progeny circle from a parental circle. An important question is whether the second of 2 DNA replication proceeds in the same manner as the first round of replication or whether it involves a different process. To answer this question, we have carried out the following experiments. Bacteria infected with [~HJ-labelled ,~ phage were incubated to allow 2 DNA replication in heavy medium at 37°C (or 32°C) for 10 rain, 15 rain, 20 rain, 30 rain and 50 rain. 2 DNA was extracted and purified by CsC1 eentrifugation to investigate the fate of parental ~ DNA. Fifteen rain after infection of E. eoli by 2elIclII, for example, about 36% of the label in parental DNA was found in a half-heavy position, indicating that parental 2 DNA had started to replicate (Fig. 2A). Electron microscopic observations on the replicating molecules located between the density of light and half-heavy peaks exhibited predominantly (71%) double-branched 0-type replicating structures. This result is quite consistent with the previous observations that, after 10 rain of infection, 85% of the replicating molecules exhibited a double-branched circular structure (Ogawa et al., 1968; Schn6s and Inman, 1970). (Ten rain sample of 2 c l I c l I I exhibited 83% of 0-form as shown in Table 1.) Thirty rain after infection, the progeny circles located at the half-heavy position had started to replicate and we have observed replicating molecules in the fractions between the half-heavy and full-heavy positions. We observed that almost all (about 99%) of these replicating molecules exhibited a single-branched rolling-circle (Sigma ((~)-type) structure despite the fact that the DNA extraction procedure was identical for the early and late samples. Fifty min after infection, the

Rolling-circle of ~ DNA

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Fig. 2A--C. Density-gradient centrifugation of A DNAs from infected cells grovax in heavy medium. E. cell was cultured in a heavy medium at a cell density of 2--~3 × 10S/ml. [SH] labelled ~ phage infected at a multiplicity of infection of 10. DNA was extracted as described in the Methods and subjected two successive CsC1 density gradient centrifugation. The positions where the heavy (HH), the half-heavy (HL) and the light (LL) DNAs are indicated with the arrows (based on the density). Fifteen rain after infection (A), 30 rain after infection and 50 rain after infection (C) of ,~clIclIl experiment. The recovery of radioactivity was about 93 %

p a r e n t a l D N A label was locate a t a still denser p o s i t i o n in t h e CsC1 g r a d i e n t a n d replicating molecules were also rolling-circle structures. W e h a v e e x a m i n e d t h e replicarive s t r u c t u r e using various 2 strains as shown in T a b l e 1. All of these e x p e r i m e n t s clearly e x h i b i t e d m o l e c u l a r t r a n s i t i o n of A R F from 0 (early) to a (late) interm e d i a t e s . E a r l y r e p l i c a t i o n lasts for a b o u t 15 rain a n d t h e n t h e l a t e m o d e of replication commenced. These o b s e r v a t i o n s i n d i c a t e t h a t t h e r e p l i c a t i v e s t r u c t u r e in t h e second half of t h e l a t e n t p e r i o d of ~ D N A r e p l i c a t i o n is p r e d o m i n a n t l y rolling-circle. I t is i n t e r e s t i n g to n o t e here t h a t a large f r a c t i o n of ~ genome d e l e t e d in ~b221 (b2, a n d int are deleted) a n d Able 11 (b2, int, red a n d gum are deleted) is n o t essential for this R F t r a n s i t i o n ( E x p e r i m e n t 3 a n d 5 in T a b l e i).

b) Starting Point and Directions o/ Replication o[ Rolling-circle To i d e n t i f y t h e e n d of tails a n d directions of r e p l i c a t i o n of rolling-circle, we used d e n a t u r a t i o n m a p p i n g ( I n m a n , 1966). All t h e molecules a n a l y z e d b y denat u r a t i o n m a p p i n g in this s t u d y are shown in Fig. 3. I n our d a t a , circular molecules of t h e b r a n c h e d circles a r e b r o k e n a t t h e m a t u r e cohesive ends (cos) (as d e t e r m i n e d b y d e n a t u r a t i o n m a p p i n g ) in o r d e r to clarify t h e l o c a t i o n a n d d i r e c t i o n of replic a t i o n of t h e b r a n c h e d molecules.

142

S. Takahashi Table 1. Transition of 2 DNA replicative structures

Genotype of 2 and E. coli 1) ~vir in w3102

Replicative No. of replicative structures after infection ]~atio of 6/0 ( % ) Structure 10 rain 15 rain 20 rain 30 rain 50 rain Early Late 0

53

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5) 2biolIcI in E835

6) ~virOts28 in CR3~

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3 57

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17

95

20 3

51 8

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13 61

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13

82

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56

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8

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72

7

21

78 16

163 67

13 100

2 179

10 168

17

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87

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100 62

11 70

4 67

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38

94

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207 32

370 144

24 170

43 471

10 168

13

92

Six strains of ~ phage are examined in an appropriate E. coli. Several times after infection, DNA replication was terminated; two CsCI density gradient fr&ctionations of each sample are run. DNA solutions in fractions located between the light (LL) and the half-heavy (HL) or between the half-heavy and the full heavy (HH) position (see Fig. 2) arc examined by electron microscopy. Experiment (6) was carried out at 32°C (Takahashi, 1975b), all others were at 37 ° C.

As shown in Fig. 3, t h e r e p l i c a t i n g molecules which possess a linear b r a n c h e d p o r t i o n r e p l i c a t e d f r o m r i g h t t o left a t a f r e q u e n c y of 39% (33 molecules o u t of 85) a n d o t h e r 61% (52 molecules o u t of 85) r e p l i c a t e d in t h e reverse direction. These molecules f r e q u e n t l y s t a r t e d f r o m t h e r i g h t half of each molecule a n d m o v e d a w a y t o t h e left or t h e right, as t h e r e p l i c a t i o n progressed. Most f r e q u e n t l y r e p l i c a t e d region in t h e l a t e stage of ~ D N A r e p l i c a t i o n is shown b y a h i s t o g r a m in Fig. 4 A . This h i s t o g r a m indicates t h a t t h e highest f r e q u e n c y of r e p l i c a t i o n of t h e tails l o c a t e d in t h e r i g h t half of ~ D N A suggesting t h a t t h e second r o u n d of r e p l i c a t i o n h a d s t a r t e d w i t h i n a region of 16.6 ~= 5.6% from t h e r i g h t end of ~ genome. This region include a p o s t u l a t e d s t a r t i n g p o i n t of t h e first r o u n d of ~ D N A r e p l i c a t i o n (18% f r o m t h e r i g h t end) (Schn6s a n d I n m a n , 1970). I f r e p l i c a t i o n h a d s t a r t e d f r o m this region, t h e b r a n c h p o i n t (replication fork) should m o v e a w a y f r o m this p o i n t as r e p l i c a t i o n progressed. H i s t o g r a m (B) of Fig. 4 which shows t h e l o c a t i o n of b r a n c h p o i n t s d e m o n s t r a t e s this t e n d e n c y . T h e m i d p o i n t s of t h e tails of t h e r e p l i c a t i n g molecules (histogram C of Fig. 4) are m o s t f r e q u e n t l y l o c a t e d in t h e r i g h t region of 2 D N A . F r e e ends of t h e tails is also l o c a t e d m o s t f r e q u e n t l y in t h e s t a r t i n g region. This t e n d e n c y suggests t h a t t h e nicks are f r e q u e n t l y i n d u c e d in this region. T h e f a c t t h a t t h e a n t i p o d e (apo) region e x h i b i t e d r e l a t i v e l y low f r e q u e n c y of free ends suggests t h a t t h e

Rolling-circle of )LDNA

143

GENOME UNIT -2

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Fig. 3. A record of the position of branch points and free ends of the tails in the 85 replicating Based on denaturation mapping, the linear tail molecules have been aligned on the vegetative A map (genome unit 1). Tails which are longer than one unit of Z DNA are a.ligned to the adjacent next )I map (genome unit 2 or genome unit minus 1, according to the direction of replication). The free ends and branch points are shown by arrows and short vertical lines. The starting point of the first round of ADNA replication is shown by dotted lines in each A unit. Genome units are shown as 1, 2 and 3 (replication to the right) and --1 and --2 (replication to the left) from the left end of the genome unit 1. All of the molecules are arranged so that their free ends lie within genome 1

rolling-circle molecules.

t e r m i n a t i o n of the first r o u n d of replication is n o t a prerequisite to form a tailed molecules. Out of 85 replieative molecules analyzed, 26 molecules (31%) exhibited branehes with l e n g t h of more t h a n one ~ genome (Fig. 5). I t is assumed t h a t these long tailed (over one u n i t length of 2 genome) rolling-circle molecules are derived from the shorter tailed molecules (shorter t h a n one 2 length) as the replication proceeds. Seven of these longer tails (shown i n Fig. 6) appear to have b e g u n u n i d i r e c t i o n a l replication from the origin to left or to the right. If these long tailed rolling-circle molecules s t a r t e d b i d i r e e t i o n a l l y from the origin a t the b e g i n n i n g of replication

144

S. Takahashi

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Fig. 4A--D. Histograms which show the replication origin, branch points, midpoints and free ends of the tails. All the tailed molecules (including tails longer than one unit of 2 DNA) was aligned on the 2 vegetative map. (A) This histogram indicates that the right half of 2 DNA, which include the ori site of the first round of replication, was most frequently replicated. (B) The location of the branch point of the tail (replicating forks) plotted on the )l map. (C) The mid points of the tails, and (D) the free ends of the tails. As references, the origin of the first round of 2 DNA replication (ori), the physical antipode (apo) of the origin and the mid point of vegetative ;t DNA (mid) respectively are shown by dotted lines

as indicated b y the shorter tailed molecules, the growing points m i g h t meet at the antipode. However, the free ends of long tails are d i s t r i b u t e d a t m a n y sites on the m a p (except for the u p p e r 7 molecules which s t a r t e d from a region close to the

P~olling-eirele of 2 DNA

145

MICROMETERS 5

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Fig. 5. Length distribution of the tails of 85 rolling-circle RF. The distribution of tail length of replication molecules listed in Fig. 3 are shown. The parental ~ genome (circle) is normalized to a length of 17.5 micrometers (an average length of mature ]~cIIcIH DNA). The frequency of longer tails over one unit of the Agenome (as identified by denaturation mapping) was 31% (26 molecules out of 85)

origin). This result indicates that one of the replication forks of bidirectionally replicating molecules at the beginning of the second round 2 DNA replication m a y be scissored by a special mechanism during replication before the two migrating branches meet at the antipode. According to this hypothesis, the locations of the free ends of the linear molecules in Fig. 3 or 6 (shown by arrow) indicate the positions where the scission had occurred. Discussion

I t has previously been shown that the first round of 2 DNA replication (2 DNA extracted 10 min after infection at 37 ° C) started at a fixed region 18 % from the right end and proceeded bidirectionally (66%) or unidirectionally (32 %) mainly in theta (0) shaped molecules, while 15% of the replicating molecules exhibited single-branched circles (Sehn6s and Inman, 1970). We have confirmed in the present study t h a t the replicating structures derived from 1 0 ~ 1 5 min after infection were mainly (84%) double-branched theta (0)-type molecules. I n contrast to the first round of replication, however, replicating structures extracted from the late stage of 2 replication (30~-~50 min after infection) were mostly (87 % ) single-branched circles in various 2 strains. With the aid of denaturation mapping, it was found t h a t the m a n y fractions of these sigma (a)-type molecules were partially replicated bidirectionally (or asymmetric) from the ori region of replication. This suggests that, during the DNA replication, one of the replication forks m a y have been scissored by some special mechanism, leaving the other replication fork to continue to replicate until DNA synthesis was terminated experimentally. If this scission was actually induced during replication, it will result in the length from the origin to the branch point being longer than the length from the origin to the free end. Actually 57 branched molecules (76%) out of 75 fit this estimation, and 18 molecules do not fit. This is probably due to

146

S. Takahashi FRACTIONAL

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Fig. 6. An example of electron microscopic denaturation maps of the rolling-circle P~F. Twenty molecules out of 26 replicating molecules which possessed tails longer t h a n one unit length of A DNA are shown. I n the maps circles are broken a t the cohesive end site (sos), as determined b y the denaturation maps. (The molecules are normalized to the s t a n d a r d length of ZcIIcIII, 17.5 micrometers). The denaturation sites on the tail was t h e n aligned according to the corresponding denatured sites on the circles. The n u m b e r e d lines indicates the circular molecules. B r a n c h points a n d free ends of the tails are shown b y short vertical lines and b y arrows respectively

t h e d i f f e r e n c e i n r a t e of m o v e m e n t of t h e f r e e e n d s of t h e t a i l s a r e l o c a t e d a t m a n y esting to note here that many free ends g e n o m e . T h i s s u g g e s t s t h a t t h e n i c k s (or

two branches along the molecule. The s i t e s o n 2 D N A . H o w e v e r , i t is i n t e r share several common sites on the 2 scissions) are induced in some special

Rolling-circle of )~DNA

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ori

A

B

C

Fig. 7A--C. A possible model for the origin of a rolling-circle from a circular ~ DNA molecule. At the beginning of the second round of ,%])NA replication, replication starts from the ori region by bidirectional or unidirectional mode (A). Then one of the replicating forks meets a single-strand nick (or gap), releasing the daughter strand as a tail (B). Then the other intact replicating fork continues DNA replication around the circular template forming a longer tail (C)

mechanism probably depending on the unique base suquences. The free ends of seven molecules of all the long tailed molecules in Fig. 6 are located at the region close to the origin suggesting t h a t the nicks are frequently induced in the ori region. Based on these observations, 2 DNA synthesis in the second half of the latent period m a y proceed as follows: (1) The second round of replication begins mMnly at the same origin as the first round. (2) Replication of these molecules proceeds bidirectionally (or unidireetionMly) at the beginning of replication and produces (at least initially) double-branched 0-type molecules. (We have also observed these 0-type molecules at a frequency of 5 ~ 1 8 % of total R F during the late stage of 2 DNA synthesis). (3) The 0-type molecules are then converted into the rollingcircle replicative intermediates. This transition from 0 to a solely depend on a single strand nick or gap induced in the late stage of 2 DNA replication. The nick must be highly specific for the second half latent period of 2 replication, because in the early stage (10~-15 min after infection) replicating 2 DNA intermediates are primarily double-branched circles. One possibility is t h a t this kind of endonucleolytie activity might be found in the host cell (for example, endonuclease I endonuelease II, etc.). The second possibility is t h a t a part of the replication complex (replisome) of host cells with 2 genes O and P m a y cleave one strand at the replication fork [2 genes 0 and P m a y control an endonueleolytie activity (Shuster and Weisbach, 1967; Inokuchi et al., 1973)]. The third possibility is t h a t an unidentified, 2-phage-encoded endonuclease (for example, endonucleases encoded in the b2 region) might accumulate in an infected cell in the late latent period (Becket, 1970; Ando et al., 1972 ; Rhoades and Meselson, 1973 ; Freifelder et al., 1973). Ogawa and Tomizawa (1968) have reported t h a t when parental strands of 2 DNA extracted 8 rain after infection were examined in alkaline sucrose gradients, several single-strand breaks (an average of three per whole single-strand linear strand) were found. These nicks m a y accumulate in the late stage of 2 DNA replication. Similar nicking activity was investigated in T4 infected cells b y Broker (1971) ; 30 rain after infection of E. coli b y T4 (pol-. lit- double mutant),

148

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Fig. 8A D. Possible origin of a rolling circle replication intermediate. The principal assertion of the model is that the circular molecule is converted into a rolling circular replicative intermediate in the second round of Z DNA replication. Models are based on the experiment carried out in a heavy medium infected with a light phage. A thick line indicates a replicated new strand. The replicated molecule gets heavier as replication progresses from light (LL) parental strands to once replicated half-heavy (ttL) and then to molecules replicated more than once (HL-HH). Parental circles replicate from a fixed origin (ORI) mainly bidirectionally (SchnSs and Inman, 1970) and produce daughter circles. Daughter circles begin the second round of replication as shown in the model (A), (B), (C) and (D). Possible initiation events in the second round of replication and its intermediate to form the rolling-c@vle intermediates arc shown between density (HL) and (HHL). Through these possible intermediates, the "rolling-circle" intermediate is formed and produces long progeny concatemers that are probably encapsulated into a progeny phage. ~ endonucleolytic scission. × recombination

for example, the p a r e n t a l T4 D N A was e x a m i n e d b y alkaline sucrose centrifugation. Broker f o u n d one nick per 2 micrometers of T4 single-strand DNA. If similar nicks are i n d u c e d in intracellular i D N A as reported b y Ogawa a n d T o m i z a w a (1968), n e a r l y 10 single-strand breaks per whole single-strand ~ DI~A molecule would be expected. I t is also possible t h a t these gaps i n the replicating

DNA might be the result of a failure to seal the gaps in Okazaki fragments when the RNA primer is removed (Hirose, Okazaki and Tamanoi, 1973). Several mechanisms for converting a circular molecule into a rolling-circle replicative intermediate are possible. In the first model [model (A) in Fig. 8], after one round of replication which generates progeny circles, one of the strands of the progeny circle is nicked by an endonuclease before the second round replication starts, then this strand is pulled out by T4 gene 32-1ike (melting or unwinding) protein. Synthesis along the circular template begins at the 3'-OH

l~olling-circle of ~t DNA

149

terminus of the nicked strand. The linear single-strand can also be onverted into a double-strand by DNA synthesis. This branch point continues DNA synthesis using the template of circular 2~ genome producing a long linear concatemers. This type of rolling-circle was first postulated in •X-174 DNA replication (Baas and Jansz, 1972; Franke and Ray, 1972). This model required an base specific endonuclease to cut at or near gene A locus of q~X DNA. The ~X-174 gene A protein is also essential for this conversion (Johnson and Sinsheimer, 1974). I n ¢X-174, the concatemer linear DNA produced could be a single-strand because mature phage particles contain single-stranded DNA; however, 2 progeny strands must be double-stranded to be encapsuled. According to this model, the free end of the tail should always be located at the same position as the initial nick. This model does not fit our present observations, because 2 DNA replication in the second round started bidirectionally or unidirectionally from the ori region and the free ends were located at many sites on the ~ genome. In the next model [model (B) in Fig. 8], the second round of replication starts like the first round of replication; a gap or an endonucleolytic cleavage at one of the growing forks is then induced, while the other intact fork may continue to replicate, forming a long concatemer. This model requires an unknown mechanism of the release of one of the branch points from the parental circle. This breakage must be specific for the late stage of replication, because, in the first round (10-15 min after infection), 84 % of the branch points were maintained as 0-type molecules (Table I). This endonucleolytic scission may be caused by a ~-encoded endonuclease that accumulates late in the latent period. It is possible that this breakage occurs at several specific regions or sites on the ~ genomc (similar to the action of many restriction enzymes on ~ DNA, Arber, 1974). To produce long tails, it is essential to seal the once gapped sites, after releasing the tail, with ligase before the intact replication fork approaches to the gap. This sealing could be done by the host ligase (Gottesman et al., 1973). During vegetative growth, circular ~ DNA may recombine to form two circles attached at the recombination point by a cross-over. An endonuclease might convert this structure into a rolling-circle molecule [model (C) in Fig. 8]. The connected fork then act like a replicating branch point to form a long linear coneatemer. This model requires recombination proteins and an endonuclease specific for the crossing-over region. Stahl et al. (1973) proposed this model for the formation of a rolling-circle via ~ recombination gene red . I t was found that ), progeny phage production in a recombination deficient system (Rec- bacteria infected with a ~ red-) results in a small number of progeny phage. Enquist and Skalka (1973) reported that ~ DNA synthesis in a recombination deficient system is reduced to a level of about 20-50 % of that of wild type system. However, they found that long concatemer ~ DNA is found even in this recombination deficient system and that these coneatemers (as judged by sucrose gradient fraetionation and by electron microscopy) seemed normal in structure and are packed into progeny ~ heads with good efficiency. This result indicates that recombination functions (rec, red and int) m a y play some role in ~t DNA replication but that these functions are not essential for forming a rolling-circle l%F. We have recently confirmed that the rolling-circle could be formed even in a RecA-B-, int-, red-, gain- system (Furth and Takahashi, in

150

S. Takahashi

Fig. 9.

Fig. 10.

Rolling-circle of )~ DNA

151

Fig. 11.

Fig. 9. Electron micrograph of a rolling-circle replicative intermediate. The replicating daughter strand is connected a t a b r a n c h point (black arrow) with a short single-stranded region. The branch point is also characterized b y a short single-strand whisker (which is probably produced b y one of the daughter strand). This roling-circle molecule is replicated over 100% of the circular templete (the free end is shown by a white arrow). A simple circle located to the right side of the center (C) has a length of a b o u t 17.3 micrometers, and the replicating circle (C) was also a b o u t 17.3 micrometers. These molecules were found in fraction 21 of Fig. 2 Fig. 10. Electron micrograph of a double-branched t h e t a (0)-type R F with a tail. Branch points are shown b y arrows. This molecule suggests t h a t the double-branched 0-type I~:F m a y change to a single-branched a-type R F as DNA replication progresses. This molecule was located in fraction 20 of Fig. 2 Fig. 11. Electron micrograph of a double-tailed rolling-circle. Circular molecule (C) was branched with a tail (shown b y upper arrow) ; this tail is also branched (lower arrow). This doubletailed rolling-circle could originate by a second starting of replication on the tail (because the tail would possess a ori site). This molecule was found in fraction 22 of Fig. 2 5 )Iolec. gen. Genet.

152

S. Takahashi

preparation). I t is possible, however, that each recombination and replication process forms a di/ferent type of concatemer by a di//erent mechanism ; for example, the 2 recombination system can form oligomeric circular DNA by reciprocal recombination, even in a replication deficient system (Takahashi, 1975a); on the other hand, replication m a y form long concatemers by a rolling-circle. At the final step of the first round replication, the daughter strand might form a strand bridge between two progeny circles. Each branch point m a y then continue DNA synthesis to make a longer bridge [model (D) in Fig. 8]. During this gridge formation, an endonuclease might form a rolling-circle type intermediate by cutting the bridge. Forming this bridge between the two circles would require the incomplete separation of the two circles at the final step of the first round replication; one of the growing strands would also have to jump over onto the other circle. In the present study, however, we have observed no bridge between two circles; it is, however, possible that this structure might be extraordinarily unstable. We have recently carried out one simple experiment to test this model: when initiation of replication is delayed for 30 rain after infection using a 20ts m u t a n t at its nonpermissive temperature, nearly all replicating molecules are single-branched rolling-circle forms even in the /irst round replication. Thus, no termination of the circular molecules is required for the generation of singlebranched molecules. This also indicates t h a t the first rounds of 0-shaped replication could be bypassed under these conditions (Takahashi, 1975c). The most simple explanation is that these longer-tailed molecules start to replicate from the ori region bidirectionally or unidirectionally. During this round of replication (at least, before the two replicating forks meet), one of the branch points is cleaved by an endonuclease (or meet at a single-strand gap), allowing the other growing point to replicate continuously along the circular template. The long concatemer 2 DNA produced b y rolling-circle molecules m a y be the preconrsor DNA required for production of progeny phage, because the packaging of 2 DNA requires at least two cohesive sites (cos) (Feiss and Margulis, 1974; Freifelder et al., 1974). If this packaging is polarized from left to right as suggested by E m m o n s and Syvanen (personal communication), the direction of rolling-circle replication is independent from the direction of phage maturation, because we have observed t h a t the rolling-circle replicate in either direction. I t is interesting to note here t h a t bacteriophage T4 DNA replication (Bernstein, H and Bernstein, C., 1973) and ribosomal gene amplification of Xenopus oocytes (Houreade et al., 1973; Rochaix et al., 1974) also contain rolling-circle intermediates. Whether these various rolling-circle intermediates found in different organisms originate in the same manner or by different mechanisms is not clear. As proTided in the present study, the origin of 2 rolling-circle seems to be different from t h a t of the ¢ X rolling-circle (Schr6der et al., 1973).

Acknowledgements. I acknowledge the critical reading and suggestions on this manuscript contributed by Drs. R, Baldwin, R. Calendar, W. Dove, J. Geisselsoder, A. Skalka and M. Syvanen. The advice and help of Drs. D. Chattorag, I~. Inman and B. Younghusband, Mr. J. Engler and M. Furth are gratefully noted.

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References Ando, T., Mitami, M., Ohmasa, M., Itokosawa, T.: Biochim. biophys. Acta (Amst.) 269, 376-384 (1972) Arber, W.: In: Progress in nucleic acid research and molecular biology (Cohen, W.E., ed.), vol. 14, p. 1-37. 1974 Baas, P.D., Jansz, H. S.: J. molec. Biol. 63, 557-568 (1972) Becker, A. : Biochim. biophys. Res. Commun. 41, 63-70 (1970) Bernstein, H., Bernstein, C. : J. molec. Biol. 77, 355 361 (1973) Broker, T.R., Lehman, I. R. : J. molec. Biol. 60, 131-149 (1971) Cairns, J. : Cold Spr. Harb. Syrup. quant. Biol. 28, 43-45 (1963) Carter, B.J., Shaw, B.D., Smith, M. G. : Biochim. biophys. Acta (Amst.) 195,494-505 (1969) Eisen, H., Pereira de Silva, L., Jacob, F.: Cold Spr. Hath. Syrup. quant. Biol. 33, 755-764 (1968) Enquist, L.W., Skalka, A. : J. molec. Biol. 75, 183-212 (1973) Feiss, M., Margulis, T.: Molec gem Genet. 127, 285-295 (1973) Franke, B., P~ey, D. S. : Proc. nut. Acad. Sci. (Wash.) 69, 475-479 (1972) Freifelder, D., Chud, L., Levine, E.E. : J. molec. Biol. 83, 503-509 (1974) Freifelder, D., Kirschner, I., Goldstein, R., Baran, N. : J. molec. Biol. 74, 703-720 (1973) Gilbert, W., Dressier, D. : Cold Spr. I-Iarb. Syrup. quant. Biol. 33, 473-483 (1968) Gottesman, M.M., Hicks, M.L., Gellert, M.: J. molec. Biol. 77,531-547 (1973) Hirose, S., Okazaki, P~., Tamanoi, F.: J. molec. Biol. 77,501-517 (1973) Hourcade, D., Dressier, D., Wolfson, J.: Proc. nat. Acad. Sci. (Wash.) 70, 2926-2930 (1973) Inman, R.B.: J. molec. Biol. 18, 464-476 (1966) Inokuchi, H., Dove, W., Freifelder, D. : J. molec. Biol. 74, 721-727 (1973) Johnson, P.H., Sinsheimer, R. : J. molee. Biol. 83, 47-61 (1974) Kiger, J., Sinsheimer, R. : J. molec. Biol. 40, 467-579 (1969) McClure, S.C.C., Gold, M.: Virology 54, 19-27 (1973) Ogawa, T., Tomizawa, J., Fuke, M. : Proc. nat. Acad. Sei. (Wash.) 60, 861-865 (1968) Rhoades, M., Meselson, M. : J. biol. Chem. 248, 521-527 (1973) Rochaix, J.D., Bird, A., Bakken, A. : J. molec. Biol. 87, 473-487 (1974) Schn6s, M., Inman, R.B.: J. molec. Biol. 51, 61-73 (1970) SchrSder, C., Erben, E., Kaerner, H.C. : J. molec. Biol. 79,599-613 (1973) Shuster, R.C., Weisbach, A.: Nature (Lond.) 233, 852-853 (1967) Skalka, A.: In: The bacteriophage lambda (Hershey, A.D. ed.), p. 539-547. Cold Spr. tlarb. Labs., Cold Spr. Harb., N.Y. 1971 Skalka, A., Poonian, M., Bartl, P. : J. molec. Biol. 64,541-550 (1972) Smith, M. G., Skalka, A.: J. gen. Physiol. 46, No. 6, part 2, 127-142 (1966) Stahl, F.W., Chung, S., Cras~mann, J., Faulds, D., Haemel, J., Lain, S., Malone, R.E., McMilin, K.D., Nozu, Y., Siegel, J., Starthern, J., Stahl, M.: In: Virus research (Fox, C.F. and Robinson, W. S., eds.), p. 487-503. Academic Press New York and London: 1973 Takahashi, S.: Biochem. biophys. Res. Commun. 61, 607-613 (1974) Takahashi, S.: Virology 64, 319-329 (1975a) Takahashi, S.: J. molec. Biol. 94, 385-396 (1975b) Takahashi, S.: Bioehim. biophys. Acta (Amst.) 895, 306-313 (1975e) Unger, R.C., Clark, A.J.: J. molec. Biol. 70, 539-548 (1973) Young, E., Sinsheimer, R.: J. molec. Biol. 311,49-59 (1968) C o m m u n i c a t e d b y H. Ozeki Dr. Seishi Takahashi Department of Biochemistry MeGill University 3655 Drummond Street Montreal, Quebec Canada H3C 1Y6