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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

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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 syn- chronous 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 link- age between the terminal nucleotide of each parental strand and the starting nucleo- tide 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 de- naturation. 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 experi- ments. 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 concen- tration 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.
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