Examination of the Structure of DNA Labeled by H1-5Budr(H3-5Budr-DNA) Revealed That Newly Synthesized Strands Were Linked to the Pre-Existing DNA

THE INITIATION OF DNA REPLICATION IN BACILLUS SUBTILIS* BY HIROSHI YOSHIKAWA

SPACE SCIENCES LABORATORY, UNIVERSITY OF CALIFORNIA, BERKELEY Communicated by Ralph Chaney, May 1, 1967 Evidence has been provided by several investigators that the replication of chrotnosomal DNA in bacteria is initiated from a fixed point and proceeds sequentially along the chromosome.1-' Such a fixed initiation point has been shown to exist in Bacillus subtilisI and Escherichia coli.2, 5, 6 There is evidence that the reactions which initiate replication also exert a control over it.'-" The initiation point may therefore be a key structure in the control of DNA replication, but the chemical nature of the initiation point has not yet been investigated. Autoradiographs of the E. coli chromosome show it to be circular,4 but because of the limited sensitivity of the technique this did not clarify the chemical nature of the linkage between the terminus and the initiation point. The chromosome of B. subtilis was not found to be circular by autoradiography, but the possibility that chromosomal breakage occurred during the isolation process cannot be eliminated. 12 The present report describes the determination of the structure of replicating chromosomes of B. subtilis at the initiation point by taking advantage of the synchronous initiation of chromosomal replication during spore germination. Even though initiation was quite well synchronized, it was difficult to judge the exact time during germination when replication started. We found that the addition of thymidine (TdR) or 5-bromodeoxyuridine (5BUdR) to thymine-requiring spores germinating in a thymineless medium initiated synchronous sequential replication. Examination of the structure of DNA labeled by H1-5BUdR(H3-5BUdR-DNA) revealed that newly synthesized strands were linked to the pre-existing DNA. We have constructed a model of a replicating chromosome which has a covalent linkage between the terminal nucleotide of each parental strand and the starting nucleotide of each newly synthesized strand. Materials and Methods.-Strains: A derivative of B. subtilis 168 strain leu-, ind-, thy- (MY2Y1U5)13 was used throughout this investigation. His-, thy- (23YlYlU6) was used as representative of the 23 strain. Derivatives of the 168 strain, leu-, met-, ade- (MU8U5U6), leu-, met-, ade- (MU8U5U16), and leu-, met-, thr- (MU8U5U5) were used for transformation recipients.17 Media and chemicals: Sporulation and germination media for the 168 strain were described previously.1 Spores of the 23 strain were prepared in liquid Schaeffer's medium which was shaken at 370C for 5 days. For labeling cells and germinating spores 5-bromodeoxyuridine-6-H3, 9.3 c/mmole (Schwarz BioResearch, Inc.); thymidine-methyl-H3, 6.7c/mmole (New England Nuclear Corp.); and phosphoric acid-P32, carrier-free (New England Nuclear Corp.) were used. Methods: Isolation and purification of spores and subsequent germination were carried out as described previously.11 DNA was isolated from germinating spores as follows: cells were har- vested and lysed by lysozyme (1 mg/ml) in buffer A (0.1 M Tris-HCl, pH 8.2, 3 X 10-3 M MgCl2, 1 X 10-3 M 2-mercaptoethanol, 30% sucrose) for 30 min at 370C. The protoplasts obtained from this procedure were spun down and the pellet was mixed in CsCl dissolved in SSC. The final density of CsCl was 1.700. This mixture was then centrifuged at 35,000 rpm at 250C for 40-60 hr and was fractionated into approximately 70 samples. More than 95% of both the bulk and pulse-labeled DNA was extracted from germinating spores by this method. Radioactivity in the DNA was assayed by heating samples in 0.1 N NaOH at 800C for 30 min and precipitation with 10% trichloroacetic acid (TCA) at 00C. The precipitates were collected on membrane 312 Downloaded by guest on September 25, 2021 VOL. 58, 1967 BIOCHEMISTRY: H. YOSHIKAWA 313

filters (Bact-T-Flex, B-6, Carl Schleicher & Schuell, Co.), and the radioactivity was measured in a Nuclear-Chicago scintillation counter. DNA was denatured by incubation in 0.1 N NaOH at room temperature for 10 mn followed by rapid neutralization with 0.2 N HCl. A microhomogenizer (Ivan Sorvall, Inc.) was used to shear 2- to 3-ml samples in SSC with 10-20 .g carrier DNA added. Exponentially growing cells of leu-, ind-, thy- were grown in medium containing H'-5BUdR for 90 min. DNA was isolated by a modified Marmur's method."1 This labeled DNA was mixed with cold normal DNA from the same strain and was centrifuged in CsCl (p = 1.700 gm/cc) at pH 7.0 with and without denaturation. Refractive indices of CsCl solutions corresponding to peak positions of each species were determined. These were: 1.4000 = light native (LL); 1.4014 = light denatured (L); 1.4041 = hybrid native (HL); 1.4080 = heavy native (HH); and 1.4094 = heavy denatured (H). These indices were used to locate 5BUdR-DNA of unknown densities in later experiments. Experimental Results.-DNA syjnthesis after thymineless germination: The addition of TdR to thymine-requiring spores germinating in a thymineless medium initiated DNA synthesis after a lag period of less than 15 minutes.'1 When 5BUdR was used, the lag period was much longer. CsCl density profiles of DNA labeled with H3-5BUdR for various lengths of time are shown in Figures 1 and 2. The spores had previously been labeled with p32. The amounts of newly synthesized H3-5BUdR-DNA were calculated from the profiles and are shown in Table 1. It was found that 5BUdR was incorporated linearly into the DNA during the lag period, even though its over-all rate of incorporation was only approximately 5 per cent of that of TdR. The density profiles in Figures 1 and 2 show that buoyant densities of H3-5BUdR-DNA gradually increased during the first ten minutes, indicating a sequential elongation. If replication is semiconservative, DNA of an intermediate density should be Y-shaped with the two small branches composed of newly synthesized hybrid DNA. Evidence will be presented below in support of this interpretation. The existence of sequential elongation was also indicated by the mode of appear- ance of genetic markers in hybrid DNA. The adenine-16 marker was gradually replicated into hybrid DNA while no other markers, including the adenine-6 marker which is located very near adenine-16,17 were replicated within 60 minutes (Table 1). Structure of 5BUdR-DNA labeled for a short time: The intermediate density of 5BUdR-DNA labeled for a short time with H3-5BUdR (Figs. la, b, and c) could result from any of the following: (a) The dilution of 5BUdR by pool thymine in the spores. This was ruled out by showing that the density was not affected by increasing the 5BUdR concentration 100 times. (b) Newly replicated DNA might be bound to proteins. This possibility was eliminated by the finding that a proteolytic enzyme (pronase) did not alter the density. (c) Incorporation of 5BUdR might represent a repair process rather than semiconservative replication. This was ruled out by the experiment shown in Figure 3. It is clear that the density of most of the 5BUdR-DNA became changed in a stepwise manner to that of hybrid DNA by hydrodynamic shearing. Shearing at 80 volts for prolonged times eventually converted more than 90 per cent of the 5BUdR-DNA into a hybrid molecule. The average molecular weight of DNA sheared at 80 volts was 2.3 X 106 daltons and at 60 volts was 4.2 X 106 daltons. The results suggested that 5BUdR was incorporated in a semiconservative manner. This was further supported by the observation that alkali denaturation of the Downloaded by guest on September 25, 2021 314 BIOCHEMISTRY: H. YOSHIKAWA PROC. N. A. S.

p32, cpm H3, cPm

p32

LHI' 400 b ILL 200

3 200 100

C'O.... .O..O-0 0

200 35 40 Tube number FIG. 1.-Density profiles of H3-5BUdR- DNA labeled for varied lengths of time. 100 P32-labeled spores of leu-, ind-, thy- at 260 Klett units were germinated for 4 hr at 370C in 75 ml of thymineless medium. HL- 5BUdR was added to produce a final concentration of 5 ,uc/ml and 0.2 Mg/ml. Aliquots 30 35 of 20 ml were incubated, and incorporation was stopped by pouring into 5 vol of iced Tube number buffer A with 2 X 10-2 M NaN3. DNA was extracted and centrifuged at 35,000 rpm for FIG. 2.-Density profiles of H3-5BUdR-DNA 60 hr in CsCl (p = 1.700 gm/cc) at 250C. (a) labeled for varied lengths of time. Experi- 1-min pulse; (b) 2 min; (c) 4 min. LL and mental conditions were the same as in Fig. 1, LH had refractive indices which corresponded except the concentration of 5BUdR was 2.0 pg/ to light and hybrid native DNA. ml. (a) 10 min; (b) 30 min; (c) 60 min. TABLE 1 SEQUENTIAL CHROMOSOMAL REPLICATION WITH 5BUdR Activity of Hybrid DNAt I-llrTMTYS- Ir. EATIrr ^Transforming Time of incubation 5BUdR-DNA Total Transforming Activity with HI-5BUdR Total DNA* (%) _ (min) (%) (Ade-16)1. I .-I (Ade-6)*1 I _ (Thr) (Met) 1 0.08 2 0.14 4 0.36 10 0.60 0.16 0.05 30 1.40 3.40 0.06 60 2.70 14.50 0.26 0.32 0.10 Experimental conditions are described in legend to Fig. 1. * Amount of 5BUdR was calculated from the total H'-activity obtained by integrating density profiles shown in Figs. 1 and 2. Total DNA was calculated from the total P32-activity in the same profiles. t Fractionated samples of Fig. 2 were assayed for transforming activity using leu-, met-, ade-16- leu-, met-, ade-6 -; and leu-, met-, thr- as recipients. Transforming 49civity Qf hybrid and light DNA regions were separately integrated and the ratio was calculated, Downloaded by guest on September 25, 2021 VOL. 58, 1967 BIOCHEMISTRY: H. YOSHIKAWA 315

O.D. (260 mit) H 3, cPm 0.4 I00 a

0.240

H3j i O.D. 0.4 0 L IL

0.2 200 FIG. 3.-Shearing effect on buoyant density of 5BUdR-DNA pulsed for 30 sec. Leu-, ind-, thy- spores were _ V.-\ germinated in thymineless medium for 0 * * 0 4 hr and pulsed with H3-5BUdR, 5 sc/ 0.4 IL 400 ml and 0.2 ,ug/ml, for 30 sec. DNA LHI was extracted in CsCl (see Methods). _ c It was dialyzed against SSC and condensed in polyethyleneglycol 6000. Carrier DNA was added and samples 0.2 H3 O.D. 200 were divided into three portions: (1) no further treatment (a); (2) sheared _1 at 60 V for 10 min (b); and (3) sheared/ at 80 v for 10 min (c). All samples were centrifuged as showninFig. 1. 0 lo 15 20 25 30 35 40 45 Tube number sheared molecules produced H3-5BUdR-DNA of a density corresponding to that of a denatured DNA strand which was fully labeled with 5BUdR (Fig. 4). On the other hand, denaturation of H3-5BUdR-DNA without hydrodynamic shearing gave a completely different result. As shown in Figure 5, density of H3-5BUdR-DNA after denaturation was heterogenous, with the major portion having a density only slightly greater than that of a light-denatured strand. This indicated that newly synthesized 5BUdR-DNA strands are covalently joined to normal thymine-DNA strands of various lengths. It was also found that the molecular weight of alkali-denatured 5BUdR-DNA was 5-10 times greater than that of denatured heavy 5BUdR-DNA which had been sheared before denaturation. DNA synthesis during thymineless germination: Two possibilities could account for the joining of a newly synthesized 5BUdR-DNA strand to a normal one. It could be that DNA replication took place by utilizing pool thymine during thymineless germination before 5BUdR was added. To examine this possibility, p32- incorporation was measured during thymineless germination (Table 2). p32 was incorporated in the DNA in varying amounts depending on the length of incubation in thymineless medium or on the age of spores. In all experiments, densities of alkali-denatured H3-5BUdR-DNA strands were the same and were independent of the amount of p32 incorporated. These results suggest that p32 incorporated during thymine starvation is not located only at the initiation point but randomly throughout the entire chromosome, possibly through a repair mechanism. To test Downloaded by guest on September 25, 2021 316 BIOCHEMISTRY: H. YOSHIKAWA PROC. N. A. S.

O.D. (260 miu) H3, cpm

10 20 30 40 50 Tube number FIG. 4.-Alkali denaturation of sheared 5BUdR-DNA. The pulse-labeled DNA sample was prepared as described in Fig. 3. It was then sheared at 80 v for 15 min and divided into two parts: (1) no further treatment (a) and (2) denatured by alkali and additional carrier added after neutralization (b). They were then centrifuged for 60 hr at 250C in CsCl (p = 1.700 gm/cc). L and H had refractive indices which corresponded, respectively, to light-denatured and heavy-denatured DNA.

this hypothesis, the spores were labeled with H3-5BUdR for one minute after germination in a thymineless medium containing p32. Alkali-denatured DNA isolated from these spores had H3 and P32 activities which were completely separable from each other in a CsCl density gradient (Fig. 6). Since these activities were separate, the intermediate density of the H3-5BUdR strands cannot be explained by the joining of 5BUdR strands to light-DNA strands which were synthesized during thymineless germination. These results support the possibility that newly synthesized DNA strands are covalently bonded to preexisting DNA strands.

TABLE 2 p32 INCORPORATION DURING THYMINELESS GERMINATION Time in thymine- H3-5BUdR/30 sec P39 less medium Total DNA Total DNA (min) Cpm (%) (Cpm) (%) New spores 135 175 0.007 1425 0.04 165 2006 0.110 2015 0.06 195 2559 0.140 3049 0.09 240 2428 0.140 3815 0.12 Old spores 240 2185 0.120 7643 0.23 Spores were germinated in thymineless medium with P32, 150 mc/mmole, for indicated time, then pulse- labeled with EL8-5BUdR for 30 sec as described in Fig. 6. Total amounts of P'- and Hs-activity were measured from the profiles which were similar to those shown in Fig. 5a. Total DNA was measured from integration of the optical density of the samples fractionated in CsCl gradients. New spores were kept in H20 at 40C for less than 2 weeks after harvesting. Old spores were stored at 40C in H20 for 3 months. Downloaded by guest on September 25, 2021 VOL. 58, 1967 BIOCHEMISTRY: H. YOSHIKAWA 317

O.D. (260 mju) H3, cpm P32 0.6 | | | - 600 _ a H{ Li ILL 200-

0.4 - 4004

0.2 -/31 00 H A/ D. 20 p32

0 ~0 0 0.6.L L 600 _ L b HHI I ILL 200

0.4 -p32 400 L'2.I*~\if.-!*\/\ */\/\ CD. 100 0.2 /-\/200

0 L... 9 0 1 10 20 30 40 50 Tube number FIG. 5-Alkali denaturation of unsheared 5BUdR-DNA. The DNA samples were the same as those described in Fig. la and c. (a) 1-min pulse-labeled DNA collected from the fractionated samples shown in Fig. la, was dialyzed against SSC. condensed, and carrier DNA was added. It was alkali-denatured. Additional carrier DNA was added and cen- trifugation was in CsCl (see Fig. 4). (b) 4-min pulse-labeled DNA was collected from the fractionated samples shown in Fig. lc and was treated as (a) above. The model of a replicating chromosome: It is possible that the chromosomes in spores are not in a complete form but are partially replicated near the initiation point. It has been reported that stationary phase cells of the 168 strain do not contain completed chromosomes, while those of the 23 strain do.22 Germinating spores of the 23 strain showed the same pattern of 5BUdR incorporation as 168 spores did. It is therefore postulated that newly synthesized DNA strands are covalently joined to a terminus of two strands of the parental chromosome (Fig. 7). In this model, initiation of a new replication cycle is an extension of the terminal nucleotide of the old strand by addition of a new deoxyribonucleotide through formation of a phosphodiester bond. This model imposes several characteristics on the structure of the replicating chromosome. (a) The replicating chromosome is circular and covalently linked at the initiation point. (b) When replication is completed, the two daughter chromosomes are joined together and their separation requires breakage of two phosphodiester bonds. This separation may automatically initiate a new replication cycle. (c) Breakage of one of the phosphodiester bonds gives rise to a dimeric chromosome joined by a single strand. Chromosomes such as these have been found in replicating phages.14' 15 Discussion.-Evidence is presented which indicates that the addition of 5BUdR to Downloaded by guest on September 25, 2021 318 BIOCHEMISTRY: H. YOSHIKAWA PRoc. N. A. S.

O.D. (260 ms) H3, cpm P32 {LH {LL 0.4 a A800 400

H3 0.2 /I 400 200 ,/ p32 D 0 * O . D O. 0

0.4H b ~Calculated p321 L40 0

0.2 H ~~ / K"O.D* 200 200

Pi 0 ~ ~~~~~~~~~~~~0 20 30 40 50 Tube number FIG. 6.-Incorporation of p32 during thymineless germination. Twenty ml of leu, ind , thy spores of 260 Klett units were germinated in thymineless medium with p32, 50 mc/mmole and 1 X 10-3 M, for 4 hr. Spores were then filtered, washed, and resuspended in the same medium with 10-2 M phosphate. After 10 min incubation at 370C, H3- 5BUdR, 5 1&c/ml, and 0.2 lAg/ml, was added for 1 min. DNA was extracted as described in Methods. Carrier DNA was added and the samples were divided into two parts: (1) no further treatment (a); (2) denatured by alkali and additional carrier added after neutralization (b). Both samples were centrifuged for 60 hr at 250C in CsCl (p = 1.700 gm/cc). The theoretical P32-activity in (b) was calculated assuming that the density of alkali-denatured DNA was determined by the ratio of P32-thymine strand to H3-5BUdR strand, so that pH - pX/pX - pL = L/H, where pH = density of heavy-denatured strand, pX = density of unknown denatured strand, pL = density of denatured light strand, and L/H = ratio of light strand to heavy strand in unknown samples.

spores germinating in a thymineless medium initiated synchronous sequential replication. This made it possible to control exactly the timing of initiation and to study the structure of a replicating chromosome near the initiation point. In this thymineless germination there was no evidence of lysis or thymineless death for 20 hours as shown by optical density and viable count. Linkage of newly replicated DNA strands to pre-existing strands was shown by measuring the density of H3-5BUdR-labeled DNA which had been subjected to alkali denaturation, or shearing, or both. The linkage is end-to-end since alkali denaturation of sheared DNA produced only heavy strands. It was assumed that newly synthesized strands are covalently linked to terminal nucleotides of two strands of the parental chromosomes. It is considered unlikely that both 168 and 23 strains have chromosomes in their spores which are partially replicated at the initiation region. The proposed model assumes circularity of the replicating chromosome of B. Downloaded by guest on September 25, 2021 VOL. 58, 1967 BIOCHEMISTRY: H. YOSHIKAWA 319

FIG. 7.-Proposed model of the Z A replicating chromosome. A = Gene near starting point. Z = Gene near z terminus. During replication, the two A ends are closed covalentlv. In se- 0 quence: structure at initiation point, partially replicated chromosome, completed chromosome, and dimeric

chromosome. This should be con- A ZIAZ Z trasted with the model proposed by A--- - Cairns.4 I Single break subtilis. Transformation and transduction have so far indicated no evidence of circularity.16 As shown in Figure 7, DNA isolated from such joined regions should have a Y-shaped structure similar to that found at the growing point.'8-2' Such Y-shaped forms might be rejected during transformation or prophage induction. These possibilities should be tested through incorporation of pulse-labeled DNA into competent cells or induced phages. The author is grateful to Miss Estelle Cook and Mrs. Barbara Jansen for their technical assist- ance, and to Dr. Thomas H. Jukes for his interest and support of this investigation. Abbreviations: Thy , ind-, leu., ade-, met , thr- refer to mutants requiring thymine, indole, leucine, adenine, methionine, and threonine, respectively. SSC, standard saline citrate (0.15 M NaCl and 0.015 M Na citrate, pH 7.0). * This work was supported by grant NsG 479 from the National Aeronautics and Space Ad- ministration to the University of California, Berkeley. 1 Yoshikawa, H., and N. Sueoka, these PROCEEDINGS, 49, 559 (1963). 2 Nagata, T., these PROCEEDINGS, 49, 551 (1963). I Bonhoeffer, F., and A. Giere, J. Mol. Biol., 7, 534 (1963). 4 Cairns, J., in Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 43. 6 Lark, K. G., T. Repko, and E. J. Hoffman, Biochim. Biophys. Acta, 76, 9 (1963). 6 Donachie, W. D., and M. Masters, Genet. Res. Camb., 8, 119 (1966). 7 Maaloe, O., in Cold Spring Harbor Symposia on Quantitative Biology, vol. 26 (1961), p. 45. 8 Lark, C., and K. G. Lark, J. Mol. Biol., 10, 120 (1964). 9 Jacob, F., S. Brenner, and F. Cuzin, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 329. 10 Kohiyama, M., H. Lanfrom, S. Brenner, and F. Jacob, Compt. Rend., 257, 1979 (1963). 11Yoshikawa, H., these PROCEEDINGS, 53, 1476 (1965). 12 Dennis, E. S., and R. G. Wake, J. Mol. Biol., 15, 435 (1966). '3 Farmer, J. L., and F. Rothman, J. Bacteriol., 89, 262 (1965). Original thymine-requiring mutants of 168 and 23 strains were kindly given by Dr. F. Rothman. 14 Frankel, F. R., J. Mol. Biol., 18, 144 (1966). 16 Smith, M. G., and A. Skalka, J. Gen. Physiol., 49, 127 (1966). 16 Yoshikawa, H., Genetics, 54, 1201 (1966). 17 Oishi, M., A. Oishi, and N. Sueoka, these PROCEEDINGS, 55, 1095 (1966). 18 Hanawalt, P. C., and D. S. Ray, these PROCEEDINGS, 52, 125 (1964). 19 Rolfe, R., these PROCEEDINGS, 49, 386 (1963). 20 Rosenberg, B. H., and L. F. Cavalieri, these PROCEEDINGS, 51, 826 (1964). 21 Kidson, C., J. Mol. Biol., 17, 1 (1966). 22 Sueoka, N., and H. Yoshikawa, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 47. Downloaded by guest on September 25, 2021