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Membrane attachment of the replication origins of a multifork (dichotomous) chromosome in Bacillus subtilis
6. Mol. Biol. (1972) 69, 237-248
Membrane Attachment Multifork (Dichotomous) M. AIDEEN
of the Replication Origins of a Chromosome in Bacillus subtilis
O'SULLIVANANDNOBORTJSUEOKA
Department of Biochemical Sciences,Princeton University Princeton, N.J. 08540, U.S.A. (Received1 November 1971) Association between the membrane and replication origins of the Badlm mbtilis chromosome has been examined during synchronous dichotomous replication where two successive initiations result in four copies of the replication origin and one copy of the terminus per chromosome. The four origins can be distinguished from each other by a combination of density transfer experiments, pulse label at the origin and hybridization with the separated strands of B. subtilk DNA. The data indicate that, when the chromosome contains four replication origins, all four are associated with the membrane.
1. Introduction The possibility of the attachment of the bacterial chromosomeon the cell membrane wa,sraised by Jacob, Brenner & Cuzin (1963) and some experimental support was provided by the electron-microscopic observations of Ryter & Jacob (1964). Subsequently, in both Bacillus subtilis (Ganesan & Lederberg, 1965) and Escherichia wli (Smith & Hanawalt, 1967) membrane binding of the replication point was suggested by a transient enrichment of pulse-labeled [3H]thymidine in the small fraction of DNA, which was found with the membrane fraction. Another type of membranechromosome association was discovered in B. subtilis, when it was shown that the replica#tionorigin and the terminus were permanently associatedwith the membrane fraction (Sueoka & Quinn, 1968). One type of evidence for this was an enrichment in the membrane fraction of genetic markers close to the replication origin and the terminus when compared with internal markers, using exponentially growing cells; another was the observation that a pulse of [3H]thymidine at the replication origin remained in the membrane fraction after a chaseby non-radioactive thymidine. The above conclusion has been strengthened by evidence from other laboratories (Snyder & Young, 1969; Fielding & Fox, 1970; Ivarie & Pene, 1970). Under certain growth conditions, replication of the chromosomeof B. subtilis and that of E. coli can be reinitiated at the chromosomereplication origin during a replication cycle (Yoshikawa, O’Sullivan & Sueoka, 1964; Oishi, Yoshikawa & Sueoka, 1964; Bird & Lark, 1968; Caro & Berg, 1969). This reinitiation is symmetric, i.e. it occurs at the origin of both branches of the replicating chromosome(Quinn & Sueoka, 1970; Fritsoh & Worcel, 1971). This type of multifork symmetric replication has been termed “dichotomous replication” (Yoshikawa et al., 1964). A chromosomereplicating in a dichotomous manner as a consequenceof two rounds of initiation will contain four copies of the replication origin and one copy of the terminus. 237
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The dichotomous system can be utilized to investigate the extent of associatiull between the membrane and the multiple origins. The results of such an invostigai8ion should, in turn, help in designing experiments to elucidate tho nature of the unique replication origin, the segregation of chromosomes and the topology of the terrnitsus-origin junction. These questions are intimately related in the following way. If the segregation of chromosomes is due directly to binding between each segregating chromosomal unit and a segregation region of the membrane, then all segregating units should be membrane associated. If each replication origin is always membmne associated, then membrane association may contribute to the unique nature of the origin site on the chromosome, and its recognition by the cell. If, however, a second initiation at the origin were to displace existing origins on the membrane, neither ot the postulates above would be true. As a first step in this investigation we have examined the membrane association of each of the four origins in a chromosome replicating in a dichotomous manner. The results of our experiments indicate that all four replication origins are associated wit’h the membrane under conditions where the replication points are far removed from the origin.
This
supports
the
idea
that
the
association
is maintained
some replication and suggests that the membrane t’ional and structural properties of the origin.
2. Materials
association
throughout
is a part
chromo-
of the func-
and Methods
(a) Spore preparation [14C]Thymine spores were prepared 4 days growth on tryptose/blood pg/ml.) and [14C]thymine (0.075 Bromouracil germination medium [3H]bromouracil (2 pCi/ml., New was terminated by the addition of
from B. subtilis 23 thyAis- (from I?. Rothman) bJ agar (Difco) plates supplemented with thymine (5 $X/ml., New England Nuclear Corp., 0.036 rg/&i). (O’Sullivan & Sueoka, 1967) was supplemented with England Nuclear Corp., 0.0181 mg/mCi). Replication KCN (0.02 M final concentration). (b)
The cells were centrifuged, resuspended and incubated with egg white lysozyme subsequently with sodium lauryl sulfate (c)
Gentle
lysis
and
Complete
lysis
in 0.05 ml. of 0.15 M-NaCl, (Worthington, 1 mg/ml.) for (2%) plus pronase (1 mg/ml.)
preparation
of membrane-bound
0.1 M-EDTA, pH 8, 60 min at 37”C, and at 37°C for 30 min.
DNA
Cells were resuspended in 0.3 ml. of TESE buffer (0.05 M-Tris, 0.05, M-EDTA, 0.05 nrEGTA, 0.5 M-sucrose, pH 8). Lysozyme was added (1 mg/ml.) and the cells were incubated without shaking (60 min, 37’C). The resultant protoplasts were burst by the addition of 1.56 ml. TES buffer (0.05 M-Tris, 0.1 M-EDT& 0.15 M-sucrose, pH 8) and sheared by mixing on a Vortex genie mixer for 2 min at full speed. The lysate was diluted by addition of 1 ml. of water and layered onto a 25-ml. sucrose gradient (5 to 20%), or onto 20% sucrose over a 5-ml. 62% sucrose shelf (Smith & Hanawalt, 1967). The gradient was centrifuged for 1 hr at 25,000 rev./min on a Spinco model L ultracentrifuge at 4°C. Fractions (15 or 30 drops) were collected from the bottom of the tube, in the cold room. Portions of 0.2 ml. were removed for alkali-resistant, acid-precipitable count, which was obtained by the addition of NaOH (1 N, 2 hr, 37”C), neutralization, precipitation and washing with cold 10% trichloroacetic acid in the presence of crude salmon-sperm carrier DNA. Samples were counted in a liquid scintillation counter. The remainder of the fractions were combined into 2 fractions, membrane-bound and free, and frozen. (d) Membrane-bound the freezer and
and brought
Cesium
chloride
gradients
free DNA fractions from the sucrose gradient were removed to 37°C. A 1 -ml. portion from both fractions was diluted with
from 2.7 ml.
MEMBRANE-ASSOCIATED
REPLICATION
ORIGINS
239
Tris/EDTA buffer (0.01 M-Tris, 0.001 M-EDTA, pH 8) to lower the sucrose concentration. The samples were incubated with pronase (100 pg/ml.) for 30 mm at 37°C. Sodium lauryl sulfate was added (0.1 o/O, final concentration) and incubation at 37°C was continued for a further 60 min. CsCl was added to both samples and to the completely lysed control to bring the final density to 1.7 g/cm3. The samples were centrifuged at 25°C for 4 days in a SW39 rotor of Spinco model 2 ultracentrifuge at 35,000 rev./mm One-drop fractions were collected from the bottom of the tube. One ml. sterile standard saline citrate (0.15 M-NaCl, 0.016 m-sodium citrate, pH 7.0) was added to each fraction. Radioactive count and transformation profiles were obtained as described previously (O’Sullivan & Sueoka, 1967). Recipient cells for transformation assays were: 168 EeuFl/met5/adelG (Mu8u5ulG) ; 168 le&/met5/ade6 (Mu8u5u6).
(e) Separation
of
light and heavy strands of DNA
Highly purified DNA was prepared from cells of B. subtilis (Marmur, 1961), denatured with alkali, neutralized, dialyzed against 0.4 M-Nacl, 0.05 M-sodium phosphate buffer, pH 6.7, and fractionated into light and heavy strands on an interrupted methylated albumin/ kieselguhr column (Rudner, Karkas & Chargaff, 1968). Five-ml. portions were collected. Analysis by CsCl density-gradient centrifugation in a Spinco model E, using 15N-labeled Pseudomonus ueruginosa DNA as a reference (density 1.743 g/cm3) showed that the densities of the two peaks were 1.719 g/cm3 (light strand) and 1.721 g/cm3 (heavy strand) and that in cross annealing (4 M-NaCl, 4 hr, 65°C) they yielded a single peak of DNA with a deusity of 1.708 g/cm3, which is somewhat denser than native DNA (1.703 g/cm3). Selfannealing of a sample from the light-strand fraction did not alter the sharpness or density of the band (1.719 g/cm3), whereas self-annealing of the heavy strand resulted in broadening of the band at the same density position (1,722 g/cm3) and the appearance of a shoulder at a density position (l-710 g/cm”) closer to that of native DNA (1.703 g/cm3). Analysis on an hydroxyapatite column showed that self-annealing of the heavy-strand fraction produced 81 y. denatured DNA and 19% “native” DNA. For hybridization 9 DNA filters were prepared (Gillespie & Spiegelman, 1965) from O.&ml. portions from methylated albumin/ kieselguhr column eluents. The filters were coded by nicking the edge.
3. Results An enrichment in membrane-associatedDNA of genetic markers near the replication origin and terminus of B. subtilis has been observed during dichotomous replication (Sueoka & Bell, manuscript in preparation). When an exponentially growing culture in Penassay (Difco) medium was gently lysed and centrifuged in a sucrose gradient over a 62% sucroseshelf, the DNA which co-sedimentedwith membranous material was enriched for origin and terminal markers, compared with internal markers. This enrichment suggesteda permanent association between all replication origins and the membrane. We have examined the replication origin-membrane associations using density transfer experiments during synchronous replication from the origin. Two synchronous systems were used: (a) germinating spores; and (b) a temperature-sensitive mutant (ha-l) for initiation of DNA replication (White & Sueoka, manuscript in preparation). In both systems media which permitted dichotomous replication were used. Replication was terminated when both first and secondrounds of replication had passedthe genetic marker adel6, which has been located closeto the replication origin (OSullivan & Sueoka, 1967). The location of this marker in relation to the replication origin and to other markers usedin this analysis is shown in a linear representation of the genetic map (Fig. l(a)). This marker has been found to show the greatest enrichment with respect to other markers in membrane-associatedDNA (Sueoka & Quinn, 1968; Snyder & Young, 1969). It is located approximately 2% or 60 x lo6 daltons
M.
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away from the origin (O’Sullivan & Sueoka, 1967). Therefore, it is not sufficiently close to the origin to be found exclusively in the membrane fraction; thus, in a gentle lysate sheared by a vortex, and having an average molecular weight of 30 x lo6 daltons, only higher molecular-weight molecules will carry this gene. Figure l(b) shows the experimental protocol used in the experiments; parental DNA is labeled with [l*C]thym ine and newly replicated DNA with 3H-labeled 5-bromouracil. Figure l(c) shows the desired density distribution of replication origins, replication points and radioactivity. The diagram illustrates that DNA carrying the ade16 marker is expected to distribute equally between heavy and hybrid density regions, when the total DNA is centrifuged in a CsCl gradient. The heavy DNA (HH) has arisen from replication of the first complements of parental DNA, the hybrid DNA from a second replication of the parental strands. The four replication origins can therefore be grouped experimentally into these two classes: HH, or completely “new” origins; and HL, containing one parental strand (“parental” origins). Our first experiments were to see if either class was preferentially associated with the membrane fraction. ode 16 ade6 (0)
0.2
0 Origin
Spore [‘+I
mef 5
0.4
0.6
0.8
I.0 Terminus
Replication
(b)
klt0
ftu5 t
I
+&Ura
A
+T+[3H]
Brk?
[~“t~~A-
Free DNA
ZJh‘t$z%---Membra”e-bau”d
T
DNA
(cl
/[“Cl
T
(d)
,
s’[HCIT
ade 16
FIU. 1. Di8grams
illustrating the rationale of the experiments. (a) Lineer representation of the genetia map of BaciZEw, m&i&?, showing the position of msrkers used in the analysis relative to the replioetion origin. (b) Experimental protoool. (a) Disgrem showing the expeotad density distribution resulting from the experimental protocol (b). Replication origins which are fully heavy in density (HH) are celled “new” origins; those having one new and one parental strsnd (HL density) 8re celled “parentsl” origins. (d) Diagram showing the expected density distribution of a [3H]bromouracil pulse label 8t the replication origin when chased with unkbeled bromour8oil.
MEMBRANE-ASSOCIATED
REPLICATION
ORIGINS
341
As a control experiment for the desired density distribution, a fraction of cells was completely lysed, centrifuged in CsCl and analyzed by transformation (Fig. 2, control DNA). The radioactive profile shows that heavy density DNA has been made, while much parental DNA is still unreplicated, i.e. light in density. The transformation profile shows that the replication is dichotomous, since the adeld marker is distributed equally between hybrid and heavy density regions (i.e. replicated twice), the leu8 marker is partially transferred to hybrid density (i.e. some of the leu8 markers have been replicated once) and the met5 marker (not shown) is completely in light density region (i.e. not yet replicated). A gentle lysate was prepared from the same culture used for the control experiment. When this lysate was centrifuged in a sucrose gradient over a dense sucrose shelf, 15% to 19”/$, of the total DNA was routinely found in the membrane fraction. When a portion of this fraction was centrifuged directly in a CsCl gradient more than 95% of the DNA banded on top of the gradient, indicating that the DNA was associated with some cellular complex. Treatment of the membrane-DNA complex with sodium lauryl sulfate followed by dialysis in the cold resulted in a loss of DNA. This loss was due to precipitation with sodium lauryl sulfate at a low temperature, and transformation assays indicated that the precipitation was preferential for replication point DNA rather than replication origin DNA, since the recovered DNA showed an increased ratio of adeldlleu8. When the membrane-bound DNA was completely solubilized, as described in Materials and Methods, input l*C and 3H counts were quantitatively recovered, and the adelblleu8 ratio remained unchanged. After centrifugation in CsCI, the membrane-associated DNA was analyzed by transformation to examine the distribution of ode16 in heavy and hybrid peaks. The data are shown in Figure 2 where the membrane-associated DNA is compared directly with total DNA and free DNA. The results show that adel6 transforming activity is found .in both heavy and hybrid density DNA peaks and that its distribution in membrane-associated DNA is comparable with its distribution in the total DNA. The conclusion from this experiment is that membrane-associated DNA contains either all four replication origins, or one parental origin (hybrid density) and one new origin (heavy density). In the experiment shown in Figure 2 we can use the membrane enrichment index (M), defined previously (Sueoka & Quinn, 1968), to measure the relative binding of replication origins and replication points in the membrane fraction. The calculated value for adel6/leu8 ratio of bound DNA divided by adeldlleu8 ratio of free DNA is 4.0, which is positive and high, as would be expected if the attachment of replication origins were permanent and that of replication points transient. We have performed similar experiments using the temperature-sensitive DNA initiation mutant dna-1 (White & Sueoka, manuscript in preparation) to synchronize replication. This mutant has been shown to maintain viability and transforming activity of its DNA at 45°C and, upon lowering the temperature to 35”C, to replicate sequentially from the replication origin under the experimental conditions used in this experiment (Matsushita, White & Sueoka, 1971; White & Sueoka, manuscript in preparation). The mutant was grown for several generations at 35°C in the presence of [‘4C]thymine, then diluted fivefold in a similar medium at 45°C (legend to Fig, 3). Residual DNA synthesis at 45”C, measured by incorporation of label, was completed by 25 minutes under the conditions used and the increment in DNA was approximately
242
M.
32 24
A.
O’SULLIVAN
AND
N.
SUEOKA
~-
-.-__.-
counts
rMembrane-bound
Control DNA
1I
DNA
16 t
.!;@.&@a: 16 1
24
32 Tube number
! i ! i
Tube number
Pm. 2. Density distribution of the ade16 marker in membrene-associated DNA from synchronous dichotomous chromosomes. Density distribution in CsCl gradients of the de16 transforming activity in total cell DNA, membrane-associated DNA and DNA which is not associated with the membrane (free DNA), when synohronous diohotomous replication in heavy density medium is terminated after spores have germinated for 140 min. The &al sucrose concentration of the membrane-assooiated DNA fraction was ebout 160/o. The samples were centrifuged for 4 days. Counts:3H,--~--~--;14C,-~-~-.Tra~formants:adel6,-~-~-;ade6,--~--~-; leu, *-A----A--..
MEMBRANE-ASSOCIATED
REPLICATION
ORIGINS
243
40%. AB a control, 5-bromouracil was added at 30 minutes to the cells and the 45°C incubation was continued for a further 30 minutes. At the end of the incubation, a portion was removed for complete lysis and analysis in a C&l gradient. The remaining cells were washedat 45”C, resuspendedat 35°C in medium containing 5-bromouracil and thymine, and allowed to replicate their DNA until the adel6 marker had been replicated twice (dichotomous replication). The cells were gently lysed, separated into membrane-associated DNA and free DNA fractions, which were centrifuged with CsCl and analyzed as in the preceding experiments. Analysis of the control sample showed that no density transfer of the adel6 marker took place before the cells were
Protocol
350 168 dm I G4C]T
Control
Membrane
-[14C] T t[3H] BrUra I
45” ,
30:
I
35”
60',
-bound
DNA
Free DNA Memb-
1 t + BrUra Control
rane
DNA
40
0
I:
14 Tube
18
22
26
30
34
38
number
FIG. 3. Density distribution of the c&e16 marker in membrane-e8sociated DNA from synchronous diohotomous chromosomes. Shown are CsCl gradient profiles of DNA prepared as in Fig. 2 from the temperature-sensitive initiation mutant 168 thy-ind-dna-I (White & Sueoka, manusoript in preparation). Overnight cultures of this mutant were grown et 30°C in 1.22% Penassay (Difco) supplemented with thymine (2 pg/ml.). Th e cells were uniformly labeled by growth et 35°C in C + medium supplemented with thymine (6 pg/ml.) &nd [W]thymine (55.75 @i/pmole, 1 @i/ml.). At Klett units 30, the cells were diluted into 1 vol. (30 ml.) of the same medium at 45% (non-permissive for initiation). After 30 min at 45”C, unlabeled bromouracil (60 pg/ml. final concentration) was added and the 45°C incubation was continued for a further 30 min. 20 ml. of cells were removed and lysed completely. The remaining cells were washed with minimal medium at 45°C and resuspended st 36°C in bromouracil germination medium supplemented with [3H]thymine (3 &i/ml., 0.0066 mg/mCi). Replication was terminated at 85 min by addition of KCN (0.02 x final concentration), the cells gently lysed, and separated into membrane-associated and free DNA on a sucrose gradient. Both freotions were prepared aa described in Fig. 2, and with the control sample, centrifuged in CsCl density gradients. All media have been previously described, (O’Sullivan & Sueoka, 1967). sH, --c] -- q --; I%, ---•--+-~--~; adel6 transformants, --O--O-.
returned at 35°C. Radioactivity distribution in the experimental samples,membraneassociatedDNA and free DNA, and transformation of the leu8 and met5 markers (not shown) coniirmed that the replication was dichotomous. In both samplesade16transforming activity was absent from the light density peak and distributed equally between hybrid and heavy density positions, showing that two rounds of replication had 17
244
M.
A.
O’SULLIVAN
AND
N.
SUEOKA
passed the ade16 gene. The distribution of ade16 in membrane-associated DNA is similar to its distribution in the free DNA sample. The enrichment index for adel6 in membrane-associatedDNA was 3% This result confirmed our conclusion that membrane-associatedDNA contains either all four replication origins or one parental and one “new” origin. To distinguish between these two possibilities we have used the samemutant, ha-l, to pulse-label the replication origin. A radioactive pulse at initiation of replication at the origin should label the new complementary strands of both parental strands, i.e. one strand of each polarity (seeFig. l(d)). I n a density transfer pulse-chase experiment, at reinitiation both labeled strands are themselves replicated and become heavy in density. Each of the two heavy origins contains one pulse-labeled heavy strand and one unlabeled heavy strand. The pulse-labeled strands are of opposite polarity. The two heavydensity origins can be distinguished from each other, since one contains labeled DNA which can hybridize with only one strand of Bacillus subtilis DNA, and vice versa. Strand separation can be achieved by interrupted gradient elution from a methylated albumin/kieselguhr column (Rudner et al., 1968) on the basisof a difference in pyrimidine content. For our experiments, pyrimidine-richness doesnot need to be a continuous property of one entire chromosomal strand since our pulse label is in synchronized cells. Therefore, to determine whether dichotomous membrane-associated DNA contained one or both of the two origins shown in Figure l(c) or (d) as heavy density DNA, we pulse-labeled the replication origin, chased to dichotomy, and isolated the membrane-associated DNA fraction. The labeled DNA in this fraction was then tested for its ability to hybridize with each of the two strands of B. subtilis DNA. Using the mutant, &m-l, chromosomeswere allowed to becomecompleted at 45°C. The cells were washed at 45”C, resuspended in medium without thymine for 20 minutes at 36°C to allow restoration of the initiation mechanism, then pulsed and chasedat 35°C. The sucrosegradient profile of a 15-secondpulsewith [3H]bromouracil, followed by a 30-minute chase in medium containing unlabeled bromouracil, is shown in Figure 4(a). A portion of cells lysed and analyzed by CsCland transformation assay showed that the replication was dichotomous at that time, that the first replication point had not yet reached the leu8 marker, and that the secondreplication points had mostly passedthe adel6 marker (i.e. the density distribution for adel6 transforming activity was HH, 39% ; HL, 58% ; LL, 3%). The results show that the earliest labeled DNA (l&second pulse) remains almost completely associatedwith the membrane even after reinitiation of replication (Fig. 4(a)). This result already suggeststhat both HH or new origins (seeFig. 1(d)) are membrane-associated,provided that the pulse is labeling a strand of each polarity. The 3H radioactivity obtained in such a short pulse is not sufficient for further analysis. A longer pulse (45 seconds)(Fig. 4(b)), also chased for 30 minutes, shows an enrichment for pulse label in the membrane-association DNA fraction, and the appearance of pulse label also in the free DNA. This is due to slight asynchrony in initiation (the initial incorporation rate is not linear) combined with the molecular-weight limit of membrane-associationDNA and to the occurrence of somerepair synthesis, which is found in the free DNA fraction (O’Sullivan & Sueoka, unpublished data). To obtain sufficient counts for analysis of the chased, pulse-labeledDNA remaining in the membrane-associatedDNA fraction the experiment was repeated, using high specific activity [3H]bromodeoxyuridine instead of [3H]bromouracil. As shown in
MEMBRANE-ASSOCIATED 160
REPLICATION
(a)
ORIGINS
246
1
Tube number Iha. 4. Sucrose gradient profiles of pulse-labeled DNA at the origin. DNA ~8s uniformly labeled with [14C]thymine, pulse-labeled to release DNA replicetion et the origin with [3H]bromouracil end chased to dichotomy in medium containing bromouracil. (a) 15-set pulse; (b) 45-set pulse. Cells of 168 thy-ind-&a-l were grown for several generations in C+ medium supplemented with [14C]thymine (0.2 @i/ml., 55.75 &X/~mole). The cells were diluted fivefold in to 40 ml. of the same medium et 45°C and incubated for 60 min. The cells were washed with minimal medium (80 ml.) et 46°C and resuspended in C + medium without thymine at 35°C. After 20 min the cells were placed on & Millipore filter apparatus, prewarmed to 35°C. Suction was 8pplied, [sH]bromouracil in 1 ml. C+ medium ~8s added (80 @i, 0.0079 mg/mCi), the cells washed with minimal medium (80 ml.) and resuspended in C+ medium (40 ml.) supplemented with S-bromoumcil(40 pg/ml.) end thymine (3 @i/ml.). Incubation et 36°C was continued for 30 min. A gentle lysste we8 prepared and cen3H, --a --A --. trifuged in sucrose as described in Fig. 1. 14C, me.--•.-;
Figure 5(a), the [3H]bromodeoxyuridine pulse results in enrichment for 3H counts in the membrane-associated DNA fraction, and the appearance of 3H counts also in the free DNA. Since HH and HL de16 markers are equally represented in the dichotomous membrane-associated DNA fraction (Figs 2 and 3), analyzing this DNA fraation is sufficient to examine the density-transfer order of pulse label relative to the c&16 marker (see Fig. l(d)). This eliminates the complication caused by the presence of labeled repair synthesis in the total DNA and in the free fraction. The sucrose gradient profile of this experiment is shown in Figure 5(a). The membrane-associated DNA fraction was pooled, a portion was treated with pronase and sodium lauryl sulfate and centrifuged in a CsCl gradient. The results (Fig. 5(b)) show that the membrane-associated pulse label was chased out of LL (non-replicated) density position, mainly into HH DNA. The de16 transformation profile showed that in most of the chromosomes the second replication point had passed the adeld marker. Incomplete transfer of pulse label into HH density position is most likely due to some localized asynchrony of the replication point. Residual pulse label in the LL position was 2% and was equivalent to residual transforming activity in the ade16 marker. The experiment shows that the pulse-labeled strands had mostly re-replicated (74 to 77 y. of the label was HH), whereas the aclel6 marker had been 66% re-replicated (i.e.
246
M.
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O’SULLIVAN
AND
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(b) 600 -
Tube
number
FIQ. 6. (a) Suorose gradient proiile of DNA pulse-labeled and chased. The experiment is the same aa that described in Figure 4, except that the pulse was performed with [3H]bromodeoxyuridine (2 &i/ml., 0.0326 mg/mCi) for 10 sec. (b) CsCl gradient profile of the membrane fraction, shown in (a), which w&a oentrifuged in a C&l gredient following the procedure described in Fig. 2. IW, -e--e--; 3H, --A --A --, ade16 transformanta, a--x -.x -..
density distribution for ade16 transforming activity was HH, 39%; HL, 59%; LL, 2%). The pulse label must therefore be at, or very close to, the replication origin, since it is re-replicated even earlier than the ade16 marker. To ensure that the pulse-labeled DNA at the replication origin represented both new strands (i.e. strands of each polarity), a hybridization experiment wss performed between pulse-labeled, chased membrane-associated DNA, and each of the separated strands of total B. subtilis DNA. The results of the hybridization experiment (Fig. 6) show that the pulse-labeled DNA can be hybridized equally with each of the two strands of DNA. The pulse label, therefore, most likely represents both DNA strands of opposite polarities. Therefore, both HH or new origins would appear to be membrane bound.
4. Discussion During dichotomous replication with two rounds of initiation, the B. subtilis chromosome contains four origins, two of which contain parental DNA strands (HL) and the other two, new DNA strands (HH) only. The results show that both types of
MEMBRANE-
ASSOCIATED
REPLICATION
ORIGINS
247
0.8
0.8
06 g :2 (u :i: 04
02
Tube number
Fro. 6. Separation of the two strands of B. mbtilie DNA and hybridization of the strands with pulse-labeled, chased, membrane-bound DNA. Total B. Bubtilia DNA was separated into light and heavy strands and 9 fraotions were immobilized on nitrocellulose Alters, as described in Materials and Methods. Pulse-labeled, ohased, membrane-bound origin DNA was obtained as described in the legend for Fig. 4, except that the pulse label was 1.5 sec. [3H]thymidine (2 Pi/ml., 0.0136 mg/mCi) and the chase medium was C+ supplemented with thymine (20 pg/ml.). The membrane fraction was diluted with 1 vol. of Tris/EDTA, treated with pronase (100 pg/ml., 30 min, 37”C), and subsequently with sarkosyl NL97 (1 O/J). NaOH wae added (1 M, final concentration) and incubation was continued for 2 hr. The solution was neutralized, dialyzed extensively and lyophilized. The DNA was dissolved in water, heated and quickly cooled and the volume adjusted to 7 ml., which contained 2 x SSC, 30% dimethylsulfoxide (Legault-DBmare, Desseaux, Heyman, S&or & Ress, 1967). The 9 DNA titers, plus a washed blank flter, were immersed in the vial containing the pulse-labeled DNA in 2 x SSC, 30% dimethylsulfoxide. Hybridization was carried out at 50°C for 18 hr with shaking, and the filters were washed according to the method of Legault-DBmare et aE. (1967), except that twioe as much of each washing solution was used. O.D. 260 nm, -a-a--; 3H ots/min hybridized, --x --x --.
replication origin are equally well associatedwith the cell membrane. They also show that our pulse-labeling technique labels new DNA strands at, or very close to, the replication origin, since the pulse-labeled DNA is re-replicated before the early genetic marker, adel6, when the replication becomesdichotomous. The hybridization experiment shows that the pulse-labeled DNA which remains associated with the membrane represents both the complementary strands. The most reasonable interpretation of these results is that both new strands of DNA made at the replication origin remain membrane-bound after they are replicated again. The results also show that reinitiation in mutant dnu-1 is symmetric, since the pulse label, representing strands of both polarities, is more than 50% transferred to heavy density before the adel6 gene is fully re-replicated (Quinn & Sueoka, 1970). Both pulse-labeled strands remain membrane bound and these represent the two origins containing no parental DNA in the dichotomous state. Since the two origins containing the parental strands are equally well bound to the membrane, we conclude that all four replication origins are associated with the membrane.
248 This work was National Institutes also acknowledge Hartford Foundation
M.
A.
O’SULLIVAN
AND
N.
SUEOKA
supported by grants from the National S&once Foundation and of Health. We thank Brenda 13. Mihan for technical assistance. the use of facilities made available by the Whitehall and John to the Department of Biology, Princeton University.
the \Vc -1.
REFERENCES Bird, R. & Lark, K. G. (1968). Cold Sp. Harb. Symp. Quant. Biol. 33, 799. Caro, L. G. & Berg, C. M. (1969). J. Mol. Biol. 45, 325. Fielding, P. & Fox, C. F. (1970). Biochem. Biophys. Res. Comm. 41, 157. Fritsch, R. & Worcel, A. (1971). J. Mol. Biol. 59, 207. Ganesan, A. T. & Lederberg, J. (1965). Biochem. Biophys. Res. Comm. 18, 824. Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, 829. Ivarie, R. D. & Pene, J. J. (1970). J. Bad. 104, 839. Jacob, F., Brenner, S. & Cuzin, F. (1963). Cold Spr. Harb. Symp. Quant. Biol. 28, 329. Leg&t-DBmare, J., Desseaux B., Heyman, T., S&or, S. & Ress, G. P. (1967). Biochem. Biophys. Res. Comm. 28, 550. Marmur, J. (1961). J. Mol. Biol. 3, 208. Matsushita, T., White, K. P. & Sueoka, N. (1971). Nature New Biology, 232, 111. Oishi, M., Yoshikawa, H. & Sueoka, N. (1964). Nature, 204, 1069. O’Sullivan, M. A. & Sueoka, N. (1967). J. Mol. Biol. 27, 349. Quinn, W. G. & Sueoka, N. (1970). Proc. Nat. Acad. Sci., Wash. 67, 717. Rudner, R., Karkas, J. D. & Chargaff, E. (1968). Proc. Nat. Acad. Sci., Wash. 60, 630. Ryter, A. & Jacob, F. (1964). Ann. Inst. Pasteur, 107, 384. Snyder, R. W. & Young, F. E. (1969). Biochem. Biophys. Res. Gomm. 35, 354. Smith, D. W. & Hanawalt, P. C. (1967). Biochim. biophys. Acta, 149, 519. Sueoka, N. & Quinn, W. G. (1968). Cold Spr. Harb. Symp. Quant. Biol. 33, 695. Yoshikawa, H., O’Sullivan, M. A. & Sueoka, N. (1964). Proc. Nat. Acad. Sci., Wash. 52, 973.
Membrane Attachment Multifork (Dichotomous) M. AIDEEN
of the Replication Origins of a Chromosome in Bacillus subtilis
O'SULLIVANANDNOBORTJSUEOKA
Department of Biochemical Sciences,Princeton University Princeton, N.J. 08540, U.S.A. (Received1 November 1971) Association between the membrane and replication origins of the Badlm mbtilis chromosome has been examined during synchronous dichotomous replication where two successive initiations result in four copies of the replication origin and one copy of the terminus per chromosome. The four origins can be distinguished from each other by a combination of density transfer experiments, pulse label at the origin and hybridization with the separated strands of B. subtilk DNA. The data indicate that, when the chromosome contains four replication origins, all four are associated with the membrane.
1. Introduction The possibility of the attachment of the bacterial chromosomeon the cell membrane wa,sraised by Jacob, Brenner & Cuzin (1963) and some experimental support was provided by the electron-microscopic observations of Ryter & Jacob (1964). Subsequently, in both Bacillus subtilis (Ganesan & Lederberg, 1965) and Escherichia wli (Smith & Hanawalt, 1967) membrane binding of the replication point was suggested by a transient enrichment of pulse-labeled [3H]thymidine in the small fraction of DNA, which was found with the membrane fraction. Another type of membranechromosome association was discovered in B. subtilis, when it was shown that the replica#tionorigin and the terminus were permanently associatedwith the membrane fraction (Sueoka & Quinn, 1968). One type of evidence for this was an enrichment in the membrane fraction of genetic markers close to the replication origin and the terminus when compared with internal markers, using exponentially growing cells; another was the observation that a pulse of [3H]thymidine at the replication origin remained in the membrane fraction after a chaseby non-radioactive thymidine. The above conclusion has been strengthened by evidence from other laboratories (Snyder & Young, 1969; Fielding & Fox, 1970; Ivarie & Pene, 1970). Under certain growth conditions, replication of the chromosomeof B. subtilis and that of E. coli can be reinitiated at the chromosomereplication origin during a replication cycle (Yoshikawa, O’Sullivan & Sueoka, 1964; Oishi, Yoshikawa & Sueoka, 1964; Bird & Lark, 1968; Caro & Berg, 1969). This reinitiation is symmetric, i.e. it occurs at the origin of both branches of the replicating chromosome(Quinn & Sueoka, 1970; Fritsoh & Worcel, 1971). This type of multifork symmetric replication has been termed “dichotomous replication” (Yoshikawa et al., 1964). A chromosomereplicating in a dichotomous manner as a consequenceof two rounds of initiation will contain four copies of the replication origin and one copy of the terminus. 237
238
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The dichotomous system can be utilized to investigate the extent of associatiull between the membrane and the multiple origins. The results of such an invostigai8ion should, in turn, help in designing experiments to elucidate tho nature of the unique replication origin, the segregation of chromosomes and the topology of the terrnitsus-origin junction. These questions are intimately related in the following way. If the segregation of chromosomes is due directly to binding between each segregating chromosomal unit and a segregation region of the membrane, then all segregating units should be membrane associated. If each replication origin is always membmne associated, then membrane association may contribute to the unique nature of the origin site on the chromosome, and its recognition by the cell. If, however, a second initiation at the origin were to displace existing origins on the membrane, neither ot the postulates above would be true. As a first step in this investigation we have examined the membrane association of each of the four origins in a chromosome replicating in a dichotomous manner. The results of our experiments indicate that all four replication origins are associated wit’h the membrane under conditions where the replication points are far removed from the origin.
This
supports
the
idea
that
the
association
is maintained
some replication and suggests that the membrane t’ional and structural properties of the origin.
2. Materials
association
throughout
is a part
chromo-
of the func-
and Methods
(a) Spore preparation [14C]Thymine spores were prepared 4 days growth on tryptose/blood pg/ml.) and [14C]thymine (0.075 Bromouracil germination medium [3H]bromouracil (2 pCi/ml., New was terminated by the addition of
from B. subtilis 23 thyAis- (from I?. Rothman) bJ agar (Difco) plates supplemented with thymine (5 $X/ml., New England Nuclear Corp., 0.036 rg/&i). (O’Sullivan & Sueoka, 1967) was supplemented with England Nuclear Corp., 0.0181 mg/mCi). Replication KCN (0.02 M final concentration). (b)
The cells were centrifuged, resuspended and incubated with egg white lysozyme subsequently with sodium lauryl sulfate (c)
Gentle
lysis
and
Complete
lysis
in 0.05 ml. of 0.15 M-NaCl, (Worthington, 1 mg/ml.) for (2%) plus pronase (1 mg/ml.)
preparation
of membrane-bound
0.1 M-EDTA, pH 8, 60 min at 37”C, and at 37°C for 30 min.
DNA
Cells were resuspended in 0.3 ml. of TESE buffer (0.05 M-Tris, 0.05, M-EDTA, 0.05 nrEGTA, 0.5 M-sucrose, pH 8). Lysozyme was added (1 mg/ml.) and the cells were incubated without shaking (60 min, 37’C). The resultant protoplasts were burst by the addition of 1.56 ml. TES buffer (0.05 M-Tris, 0.1 M-EDT& 0.15 M-sucrose, pH 8) and sheared by mixing on a Vortex genie mixer for 2 min at full speed. The lysate was diluted by addition of 1 ml. of water and layered onto a 25-ml. sucrose gradient (5 to 20%), or onto 20% sucrose over a 5-ml. 62% sucrose shelf (Smith & Hanawalt, 1967). The gradient was centrifuged for 1 hr at 25,000 rev./min on a Spinco model L ultracentrifuge at 4°C. Fractions (15 or 30 drops) were collected from the bottom of the tube, in the cold room. Portions of 0.2 ml. were removed for alkali-resistant, acid-precipitable count, which was obtained by the addition of NaOH (1 N, 2 hr, 37”C), neutralization, precipitation and washing with cold 10% trichloroacetic acid in the presence of crude salmon-sperm carrier DNA. Samples were counted in a liquid scintillation counter. The remainder of the fractions were combined into 2 fractions, membrane-bound and free, and frozen. (d) Membrane-bound the freezer and
and brought
Cesium
chloride
gradients
free DNA fractions from the sucrose gradient were removed to 37°C. A 1 -ml. portion from both fractions was diluted with
from 2.7 ml.
MEMBRANE-ASSOCIATED
REPLICATION
ORIGINS
239
Tris/EDTA buffer (0.01 M-Tris, 0.001 M-EDTA, pH 8) to lower the sucrose concentration. The samples were incubated with pronase (100 pg/ml.) for 30 mm at 37°C. Sodium lauryl sulfate was added (0.1 o/O, final concentration) and incubation at 37°C was continued for a further 60 min. CsCl was added to both samples and to the completely lysed control to bring the final density to 1.7 g/cm3. The samples were centrifuged at 25°C for 4 days in a SW39 rotor of Spinco model 2 ultracentrifuge at 35,000 rev./mm One-drop fractions were collected from the bottom of the tube. One ml. sterile standard saline citrate (0.15 M-NaCl, 0.016 m-sodium citrate, pH 7.0) was added to each fraction. Radioactive count and transformation profiles were obtained as described previously (O’Sullivan & Sueoka, 1967). Recipient cells for transformation assays were: 168 EeuFl/met5/adelG (Mu8u5ulG) ; 168 le&/met5/ade6 (Mu8u5u6).
(e) Separation
of
light and heavy strands of DNA
Highly purified DNA was prepared from cells of B. subtilis (Marmur, 1961), denatured with alkali, neutralized, dialyzed against 0.4 M-Nacl, 0.05 M-sodium phosphate buffer, pH 6.7, and fractionated into light and heavy strands on an interrupted methylated albumin/ kieselguhr column (Rudner, Karkas & Chargaff, 1968). Five-ml. portions were collected. Analysis by CsCl density-gradient centrifugation in a Spinco model E, using 15N-labeled Pseudomonus ueruginosa DNA as a reference (density 1.743 g/cm3) showed that the densities of the two peaks were 1.719 g/cm3 (light strand) and 1.721 g/cm3 (heavy strand) and that in cross annealing (4 M-NaCl, 4 hr, 65°C) they yielded a single peak of DNA with a deusity of 1.708 g/cm3, which is somewhat denser than native DNA (1.703 g/cm3). Selfannealing of a sample from the light-strand fraction did not alter the sharpness or density of the band (1.719 g/cm3), whereas self-annealing of the heavy strand resulted in broadening of the band at the same density position (1,722 g/cm3) and the appearance of a shoulder at a density position (l-710 g/cm”) closer to that of native DNA (1.703 g/cm3). Analysis on an hydroxyapatite column showed that self-annealing of the heavy-strand fraction produced 81 y. denatured DNA and 19% “native” DNA. For hybridization 9 DNA filters were prepared (Gillespie & Spiegelman, 1965) from O.&ml. portions from methylated albumin/ kieselguhr column eluents. The filters were coded by nicking the edge.
3. Results An enrichment in membrane-associatedDNA of genetic markers near the replication origin and terminus of B. subtilis has been observed during dichotomous replication (Sueoka & Bell, manuscript in preparation). When an exponentially growing culture in Penassay (Difco) medium was gently lysed and centrifuged in a sucrose gradient over a 62% sucroseshelf, the DNA which co-sedimentedwith membranous material was enriched for origin and terminal markers, compared with internal markers. This enrichment suggesteda permanent association between all replication origins and the membrane. We have examined the replication origin-membrane associations using density transfer experiments during synchronous replication from the origin. Two synchronous systems were used: (a) germinating spores; and (b) a temperature-sensitive mutant (ha-l) for initiation of DNA replication (White & Sueoka, manuscript in preparation). In both systems media which permitted dichotomous replication were used. Replication was terminated when both first and secondrounds of replication had passedthe genetic marker adel6, which has been located closeto the replication origin (OSullivan & Sueoka, 1967). The location of this marker in relation to the replication origin and to other markers usedin this analysis is shown in a linear representation of the genetic map (Fig. l(a)). This marker has been found to show the greatest enrichment with respect to other markers in membrane-associatedDNA (Sueoka & Quinn, 1968; Snyder & Young, 1969). It is located approximately 2% or 60 x lo6 daltons
M.
240
A.
O’SULLIVAN
AND
N.
SUEOKA
away from the origin (O’Sullivan & Sueoka, 1967). Therefore, it is not sufficiently close to the origin to be found exclusively in the membrane fraction; thus, in a gentle lysate sheared by a vortex, and having an average molecular weight of 30 x lo6 daltons, only higher molecular-weight molecules will carry this gene. Figure l(b) shows the experimental protocol used in the experiments; parental DNA is labeled with [l*C]thym ine and newly replicated DNA with 3H-labeled 5-bromouracil. Figure l(c) shows the desired density distribution of replication origins, replication points and radioactivity. The diagram illustrates that DNA carrying the ade16 marker is expected to distribute equally between heavy and hybrid density regions, when the total DNA is centrifuged in a CsCl gradient. The heavy DNA (HH) has arisen from replication of the first complements of parental DNA, the hybrid DNA from a second replication of the parental strands. The four replication origins can therefore be grouped experimentally into these two classes: HH, or completely “new” origins; and HL, containing one parental strand (“parental” origins). Our first experiments were to see if either class was preferentially associated with the membrane fraction. ode 16 ade6 (0)
0.2
0 Origin
Spore [‘+I
mef 5
0.4
0.6
0.8
I.0 Terminus
Replication
(b)
klt0
ftu5 t
I
+&Ura
A
+T+[3H]
Brk?
[~“t~~A-
Free DNA
ZJh‘t$z%---Membra”e-bau”d
T
DNA
(cl
/[“Cl
T
(d)
,
s’[HCIT
ade 16
FIU. 1. Di8grams
illustrating the rationale of the experiments. (a) Lineer representation of the genetia map of BaciZEw, m&i&?, showing the position of msrkers used in the analysis relative to the replioetion origin. (b) Experimental protoool. (a) Disgrem showing the expeotad density distribution resulting from the experimental protocol (b). Replication origins which are fully heavy in density (HH) are celled “new” origins; those having one new and one parental strsnd (HL density) 8re celled “parentsl” origins. (d) Diagram showing the expected density distribution of a [3H]bromouracil pulse label 8t the replication origin when chased with unkbeled bromour8oil.
MEMBRANE-ASSOCIATED
REPLICATION
ORIGINS
341
As a control experiment for the desired density distribution, a fraction of cells was completely lysed, centrifuged in CsCl and analyzed by transformation (Fig. 2, control DNA). The radioactive profile shows that heavy density DNA has been made, while much parental DNA is still unreplicated, i.e. light in density. The transformation profile shows that the replication is dichotomous, since the adeld marker is distributed equally between hybrid and heavy density regions (i.e. replicated twice), the leu8 marker is partially transferred to hybrid density (i.e. some of the leu8 markers have been replicated once) and the met5 marker (not shown) is completely in light density region (i.e. not yet replicated). A gentle lysate was prepared from the same culture used for the control experiment. When this lysate was centrifuged in a sucrose gradient over a dense sucrose shelf, 15% to 19”/$, of the total DNA was routinely found in the membrane fraction. When a portion of this fraction was centrifuged directly in a CsCl gradient more than 95% of the DNA banded on top of the gradient, indicating that the DNA was associated with some cellular complex. Treatment of the membrane-DNA complex with sodium lauryl sulfate followed by dialysis in the cold resulted in a loss of DNA. This loss was due to precipitation with sodium lauryl sulfate at a low temperature, and transformation assays indicated that the precipitation was preferential for replication point DNA rather than replication origin DNA, since the recovered DNA showed an increased ratio of adeldlleu8. When the membrane-bound DNA was completely solubilized, as described in Materials and Methods, input l*C and 3H counts were quantitatively recovered, and the adelblleu8 ratio remained unchanged. After centrifugation in CsCI, the membrane-associated DNA was analyzed by transformation to examine the distribution of ode16 in heavy and hybrid peaks. The data are shown in Figure 2 where the membrane-associated DNA is compared directly with total DNA and free DNA. The results show that adel6 transforming activity is found .in both heavy and hybrid density DNA peaks and that its distribution in membrane-associated DNA is comparable with its distribution in the total DNA. The conclusion from this experiment is that membrane-associated DNA contains either all four replication origins, or one parental origin (hybrid density) and one new origin (heavy density). In the experiment shown in Figure 2 we can use the membrane enrichment index (M), defined previously (Sueoka & Quinn, 1968), to measure the relative binding of replication origins and replication points in the membrane fraction. The calculated value for adel6/leu8 ratio of bound DNA divided by adeldlleu8 ratio of free DNA is 4.0, which is positive and high, as would be expected if the attachment of replication origins were permanent and that of replication points transient. We have performed similar experiments using the temperature-sensitive DNA initiation mutant dna-1 (White & Sueoka, manuscript in preparation) to synchronize replication. This mutant has been shown to maintain viability and transforming activity of its DNA at 45°C and, upon lowering the temperature to 35”C, to replicate sequentially from the replication origin under the experimental conditions used in this experiment (Matsushita, White & Sueoka, 1971; White & Sueoka, manuscript in preparation). The mutant was grown for several generations at 35°C in the presence of [‘4C]thymine, then diluted fivefold in a similar medium at 45°C (legend to Fig, 3). Residual DNA synthesis at 45”C, measured by incorporation of label, was completed by 25 minutes under the conditions used and the increment in DNA was approximately
242
M.
32 24
A.
O’SULLIVAN
AND
N.
SUEOKA
~-
-.-__.-
counts
rMembrane-bound
Control DNA
1I
DNA
16 t
.!;@.&@a: 16 1
24
32 Tube number
! i ! i
Tube number
Pm. 2. Density distribution of the ade16 marker in membrene-associated DNA from synchronous dichotomous chromosomes. Density distribution in CsCl gradients of the de16 transforming activity in total cell DNA, membrane-associated DNA and DNA which is not associated with the membrane (free DNA), when synohronous diohotomous replication in heavy density medium is terminated after spores have germinated for 140 min. The &al sucrose concentration of the membrane-assooiated DNA fraction was ebout 160/o. The samples were centrifuged for 4 days. Counts:3H,--~--~--;14C,-~-~-.Tra~formants:adel6,-~-~-;ade6,--~--~-; leu, *-A----A--..
MEMBRANE-ASSOCIATED
REPLICATION
ORIGINS
243
40%. AB a control, 5-bromouracil was added at 30 minutes to the cells and the 45°C incubation was continued for a further 30 minutes. At the end of the incubation, a portion was removed for complete lysis and analysis in a C&l gradient. The remaining cells were washedat 45”C, resuspendedat 35°C in medium containing 5-bromouracil and thymine, and allowed to replicate their DNA until the adel6 marker had been replicated twice (dichotomous replication). The cells were gently lysed, separated into membrane-associated DNA and free DNA fractions, which were centrifuged with CsCl and analyzed as in the preceding experiments. Analysis of the control sample showed that no density transfer of the adel6 marker took place before the cells were
Protocol
350 168 dm I G4C]T
Control
Membrane
-[14C] T t[3H] BrUra I
45” ,
30:
I
35”
60',
-bound
DNA
Free DNA Memb-
1 t + BrUra Control
rane
DNA
40
0
I:
14 Tube
18
22
26
30
34
38
number
FIG. 3. Density distribution of the c&e16 marker in membrane-e8sociated DNA from synchronous diohotomous chromosomes. Shown are CsCl gradient profiles of DNA prepared as in Fig. 2 from the temperature-sensitive initiation mutant 168 thy-ind-dna-I (White & Sueoka, manusoript in preparation). Overnight cultures of this mutant were grown et 30°C in 1.22% Penassay (Difco) supplemented with thymine (2 pg/ml.). Th e cells were uniformly labeled by growth et 35°C in C + medium supplemented with thymine (6 pg/ml.) &nd [W]thymine (55.75 @i/pmole, 1 @i/ml.). At Klett units 30, the cells were diluted into 1 vol. (30 ml.) of the same medium at 45% (non-permissive for initiation). After 30 min at 45”C, unlabeled bromouracil (60 pg/ml. final concentration) was added and the 45°C incubation was continued for a further 30 min. 20 ml. of cells were removed and lysed completely. The remaining cells were washed with minimal medium at 45°C and resuspended st 36°C in bromouracil germination medium supplemented with [3H]thymine (3 &i/ml., 0.0066 mg/mCi). Replication was terminated at 85 min by addition of KCN (0.02 x final concentration), the cells gently lysed, and separated into membrane-associated and free DNA on a sucrose gradient. Both freotions were prepared aa described in Fig. 2, and with the control sample, centrifuged in CsCl density gradients. All media have been previously described, (O’Sullivan & Sueoka, 1967). sH, --c] -- q --; I%, ---•--+-~--~; adel6 transformants, --O--O-.
returned at 35°C. Radioactivity distribution in the experimental samples,membraneassociatedDNA and free DNA, and transformation of the leu8 and met5 markers (not shown) coniirmed that the replication was dichotomous. In both samplesade16transforming activity was absent from the light density peak and distributed equally between hybrid and heavy density positions, showing that two rounds of replication had 17
244
M.
A.
O’SULLIVAN
AND
N.
SUEOKA
passed the ade16 gene. The distribution of ade16 in membrane-associated DNA is similar to its distribution in the free DNA sample. The enrichment index for adel6 in membrane-associatedDNA was 3% This result confirmed our conclusion that membrane-associatedDNA contains either all four replication origins or one parental and one “new” origin. To distinguish between these two possibilities we have used the samemutant, ha-l, to pulse-label the replication origin. A radioactive pulse at initiation of replication at the origin should label the new complementary strands of both parental strands, i.e. one strand of each polarity (seeFig. l(d)). I n a density transfer pulse-chase experiment, at reinitiation both labeled strands are themselves replicated and become heavy in density. Each of the two heavy origins contains one pulse-labeled heavy strand and one unlabeled heavy strand. The pulse-labeled strands are of opposite polarity. The two heavydensity origins can be distinguished from each other, since one contains labeled DNA which can hybridize with only one strand of Bacillus subtilis DNA, and vice versa. Strand separation can be achieved by interrupted gradient elution from a methylated albumin/kieselguhr column (Rudner et al., 1968) on the basisof a difference in pyrimidine content. For our experiments, pyrimidine-richness doesnot need to be a continuous property of one entire chromosomal strand since our pulse label is in synchronized cells. Therefore, to determine whether dichotomous membrane-associated DNA contained one or both of the two origins shown in Figure l(c) or (d) as heavy density DNA, we pulse-labeled the replication origin, chased to dichotomy, and isolated the membrane-associated DNA fraction. The labeled DNA in this fraction was then tested for its ability to hybridize with each of the two strands of B. subtilis DNA. Using the mutant, &m-l, chromosomeswere allowed to becomecompleted at 45°C. The cells were washed at 45”C, resuspended in medium without thymine for 20 minutes at 36°C to allow restoration of the initiation mechanism, then pulsed and chasedat 35°C. The sucrosegradient profile of a 15-secondpulsewith [3H]bromouracil, followed by a 30-minute chase in medium containing unlabeled bromouracil, is shown in Figure 4(a). A portion of cells lysed and analyzed by CsCland transformation assay showed that the replication was dichotomous at that time, that the first replication point had not yet reached the leu8 marker, and that the secondreplication points had mostly passedthe adel6 marker (i.e. the density distribution for adel6 transforming activity was HH, 39% ; HL, 58% ; LL, 3%). The results show that the earliest labeled DNA (l&second pulse) remains almost completely associatedwith the membrane even after reinitiation of replication (Fig. 4(a)). This result already suggeststhat both HH or new origins (seeFig. 1(d)) are membrane-associated,provided that the pulse is labeling a strand of each polarity. The 3H radioactivity obtained in such a short pulse is not sufficient for further analysis. A longer pulse (45 seconds)(Fig. 4(b)), also chased for 30 minutes, shows an enrichment for pulse label in the membrane-association DNA fraction, and the appearance of pulse label also in the free DNA. This is due to slight asynchrony in initiation (the initial incorporation rate is not linear) combined with the molecular-weight limit of membrane-associationDNA and to the occurrence of somerepair synthesis, which is found in the free DNA fraction (O’Sullivan & Sueoka, unpublished data). To obtain sufficient counts for analysis of the chased, pulse-labeledDNA remaining in the membrane-associatedDNA fraction the experiment was repeated, using high specific activity [3H]bromodeoxyuridine instead of [3H]bromouracil. As shown in
MEMBRANE-ASSOCIATED 160
REPLICATION
(a)
ORIGINS
246
1
Tube number Iha. 4. Sucrose gradient profiles of pulse-labeled DNA at the origin. DNA ~8s uniformly labeled with [14C]thymine, pulse-labeled to release DNA replicetion et the origin with [3H]bromouracil end chased to dichotomy in medium containing bromouracil. (a) 15-set pulse; (b) 45-set pulse. Cells of 168 thy-ind-&a-l were grown for several generations in C+ medium supplemented with [14C]thymine (0.2 @i/ml., 55.75 &X/~mole). The cells were diluted fivefold in to 40 ml. of the same medium et 45°C and incubated for 60 min. The cells were washed with minimal medium (80 ml.) et 46°C and resuspended in C + medium without thymine at 35°C. After 20 min the cells were placed on & Millipore filter apparatus, prewarmed to 35°C. Suction was 8pplied, [sH]bromouracil in 1 ml. C+ medium ~8s added (80 @i, 0.0079 mg/mCi), the cells washed with minimal medium (80 ml.) and resuspended in C+ medium (40 ml.) supplemented with S-bromoumcil(40 pg/ml.) end thymine (3 @i/ml.). Incubation et 36°C was continued for 30 min. A gentle lysste we8 prepared and cen3H, --a --A --. trifuged in sucrose as described in Fig. 1. 14C, me.--•.-;
Figure 5(a), the [3H]bromodeoxyuridine pulse results in enrichment for 3H counts in the membrane-associated DNA fraction, and the appearance of 3H counts also in the free DNA. Since HH and HL de16 markers are equally represented in the dichotomous membrane-associated DNA fraction (Figs 2 and 3), analyzing this DNA fraation is sufficient to examine the density-transfer order of pulse label relative to the c&16 marker (see Fig. l(d)). This eliminates the complication caused by the presence of labeled repair synthesis in the total DNA and in the free fraction. The sucrose gradient profile of this experiment is shown in Figure 5(a). The membrane-associated DNA fraction was pooled, a portion was treated with pronase and sodium lauryl sulfate and centrifuged in a CsCl gradient. The results (Fig. 5(b)) show that the membrane-associated pulse label was chased out of LL (non-replicated) density position, mainly into HH DNA. The de16 transformation profile showed that in most of the chromosomes the second replication point had passed the adeld marker. Incomplete transfer of pulse label into HH density position is most likely due to some localized asynchrony of the replication point. Residual pulse label in the LL position was 2% and was equivalent to residual transforming activity in the ade16 marker. The experiment shows that the pulse-labeled strands had mostly re-replicated (74 to 77 y. of the label was HH), whereas the aclel6 marker had been 66% re-replicated (i.e.
246
M.
A.
O’SULLIVAN
AND
N.
SUEOKA
(b) 600 -
Tube
number
FIQ. 6. (a) Suorose gradient proiile of DNA pulse-labeled and chased. The experiment is the same aa that described in Figure 4, except that the pulse was performed with [3H]bromodeoxyuridine (2 &i/ml., 0.0326 mg/mCi) for 10 sec. (b) CsCl gradient profile of the membrane fraction, shown in (a), which w&a oentrifuged in a C&l gredient following the procedure described in Fig. 2. IW, -e--e--; 3H, --A --A --, ade16 transformanta, a--x -.x -..
density distribution for ade16 transforming activity was HH, 39%; HL, 59%; LL, 2%). The pulse label must therefore be at, or very close to, the replication origin, since it is re-replicated even earlier than the ade16 marker. To ensure that the pulse-labeled DNA at the replication origin represented both new strands (i.e. strands of each polarity), a hybridization experiment wss performed between pulse-labeled, chased membrane-associated DNA, and each of the separated strands of total B. subtilis DNA. The results of the hybridization experiment (Fig. 6) show that the pulse-labeled DNA can be hybridized equally with each of the two strands of DNA. The pulse label, therefore, most likely represents both DNA strands of opposite polarities. Therefore, both HH or new origins would appear to be membrane bound.
4. Discussion During dichotomous replication with two rounds of initiation, the B. subtilis chromosome contains four origins, two of which contain parental DNA strands (HL) and the other two, new DNA strands (HH) only. The results show that both types of
MEMBRANE-
ASSOCIATED
REPLICATION
ORIGINS
247
0.8
0.8
06 g :2 (u :i: 04
02
Tube number
Fro. 6. Separation of the two strands of B. mbtilie DNA and hybridization of the strands with pulse-labeled, chased, membrane-bound DNA. Total B. Bubtilia DNA was separated into light and heavy strands and 9 fraotions were immobilized on nitrocellulose Alters, as described in Materials and Methods. Pulse-labeled, ohased, membrane-bound origin DNA was obtained as described in the legend for Fig. 4, except that the pulse label was 1.5 sec. [3H]thymidine (2 Pi/ml., 0.0136 mg/mCi) and the chase medium was C+ supplemented with thymine (20 pg/ml.). The membrane fraction was diluted with 1 vol. of Tris/EDTA, treated with pronase (100 pg/ml., 30 min, 37”C), and subsequently with sarkosyl NL97 (1 O/J). NaOH wae added (1 M, final concentration) and incubation was continued for 2 hr. The solution was neutralized, dialyzed extensively and lyophilized. The DNA was dissolved in water, heated and quickly cooled and the volume adjusted to 7 ml., which contained 2 x SSC, 30% dimethylsulfoxide (Legault-DBmare, Desseaux, Heyman, S&or & Ress, 1967). The 9 DNA titers, plus a washed blank flter, were immersed in the vial containing the pulse-labeled DNA in 2 x SSC, 30% dimethylsulfoxide. Hybridization was carried out at 50°C for 18 hr with shaking, and the filters were washed according to the method of Legault-DBmare et aE. (1967), except that twioe as much of each washing solution was used. O.D. 260 nm, -a-a--; 3H ots/min hybridized, --x --x --.
replication origin are equally well associatedwith the cell membrane. They also show that our pulse-labeling technique labels new DNA strands at, or very close to, the replication origin, since the pulse-labeled DNA is re-replicated before the early genetic marker, adel6, when the replication becomesdichotomous. The hybridization experiment shows that the pulse-labeled DNA which remains associated with the membrane represents both the complementary strands. The most reasonable interpretation of these results is that both new strands of DNA made at the replication origin remain membrane-bound after they are replicated again. The results also show that reinitiation in mutant dnu-1 is symmetric, since the pulse label, representing strands of both polarities, is more than 50% transferred to heavy density before the adel6 gene is fully re-replicated (Quinn & Sueoka, 1970). Both pulse-labeled strands remain membrane bound and these represent the two origins containing no parental DNA in the dichotomous state. Since the two origins containing the parental strands are equally well bound to the membrane, we conclude that all four replication origins are associated with the membrane.
248 This work was National Institutes also acknowledge Hartford Foundation
M.
A.
O’SULLIVAN
AND
N.
SUEOKA
supported by grants from the National S&once Foundation and of Health. We thank Brenda 13. Mihan for technical assistance. the use of facilities made available by the Whitehall and John to the Department of Biology, Princeton University.
the \Vc -1.
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