Proc. Nat. Acad. Sci. USA Vol. 68, No. 6, pp. 1296-1300, June 1971

Adenosine Triphosphate-Dependent Synthesis of Biologically Active DNA by Azide-Poisoned Bacteria (Bacillus subtilis/gene markers/detergent Brij-58) A. T. GANESAN Lt. J. P. Kennedy, Jr., Laboratories for Molecular Medicine, Department of Genetics, Stanford Medical School, Stanford, California 94305 Communicated by Joshua Lederberg, April 5, 1971

ABSTRACT Nonviable cells of Bacillus subtilis, when For characterization of the products of synthesis, cells were made permeable by treatment under controlled condi- grown in a medium containing 15NH4+, D20, and [14C]thymine tions with nonionic detergent, can be shown to replicate DNA normally and to repair certain regions of the chromo- (3) to give the DNA a buoyant density of 1.756 instead of some. The former process is stimulated by ATP in the 1.703 g/ml in CsCl. The three types of assay conditions for presence of dATP, dCTP, dGTP, and dTTP. The product, DNA polymerase (3) were (a) equimolar mixtures of the a result of semiconservative replication, is biologically deoxyribonucleosides of adenine, guanine, cytosine, and active; synthesis of the newly-formed regions of the chromosome appears to be sequential. thymine (referred to as dN) plus ATP; (b) an equimolar mixture of the four deoxyribonucleoside triphosphates Extensive biochemical and morphological evidence suggests (referred to as dNTP) of the above bases; and (c) the same as that the bacterial chromosome is replicated in an orderly (b), plus ATP. These and radioactive substrates were obtained sequential mode, possibly by an enzyme complex bound to from Schwarz BioResearch. DNA was purified carefully to cell wall membrane (1-3). Attempts to isolate a structurally minimize shear effects that might confuse repaired regions intact, active complex that would promote such a process with semiconservatively replicated sequences in density have not been very successful. Here we report a method of gradients. This was accomplished by lysing the Brij-treated making nonviable cells permeable to externally added sub- cells with sodium dodecyl sulfate (4 mg/ml) and digesting strates. These cells have lost most of their DNA polymerase the lysate with Pronase (Calbiochem) (1 mg/ml) overnight at activity and also significant amounts of nucleases and cellular room temperature (3). Cesium chloride density gradients were proteins, but are capable of continued DNA synthesis in the centrifuged in a Spinco No. 50 angle-head rotor at 34,000 rpm presence of four deoxyribonucleoside -triphosphates. This for 66 hr at 4VC. Density calculations were made with un- synthesis is stimulated by the addition of ATP. A similar labeled B. subtilis DNA of normal density as a reference; this response has been reported recently in toluene-treated cells of DNA carries mutations that do not interfere with the assay of Escherichia coli (4, 5). The Bacillus subtilis system has the the DNA species under study. Alkaline CsCl gradients were advantage that we can characterize the biological activity of used at pH 12.5 (8). In all the figures, the density positions of the product and the direction of replication. heavy, hybrid, light, and denatured heavy DNA molecules, corresponding to densities of 1.756, 1.729, 1.703, and 1.810 MATERIALS AND METHODS g/ml in CsCl are designated as HH, HL, LL, and DH. Actively growing cultures of B. subtilis, SB 168 (try-2) and SB 19 (wild type), were frozen in liquid nitrogen. These stocks RESULTS were thawed, washed, and resuspended at 109 cells/ml of 5% Live cells, when incubated with [3H]thymidine and the other sucrose solution containing 0.01 M Tris HCl buffer, pH 7.5; three deoxyribonucleosides, exhibit continued linear in- 0.001 M MgCl2; 0.001 M j3-mercaptoethanol; and sodium corporation into the DNA. However, when inhibitors such as azide, 0.05 M. These cells were then made permeable by the sodium azide are added, the incorporation stops, as is implicit addition of Brij-58 (6), a nonionic detergent. The time re- in the earlier kinetic experiments (1). During the next few quired to reach this permeable stage was monitored by ob- minutes the cells gradually lose their viability. These cells serving the cells under a phase-contrast microscope or by are not permeable to triphosphates. When such cells are staining with Giemsa (7). During a period of 1-3 hr, 55% of treated with Brij-58 and assayed for DNA synthesis in the the cellular proteins, containing more than 95% of the DNA presence of dN and ATP, incorporation is resumed after 2 hr polymerase activity [assayed with poly(dA) - poly(dT) as (Fig. la). The rate of tracer incorporation in different experi- substrate (3)], and a large portion of the nucleases were ments is similar to that observed in live cells. These Brij- released into the medium and were discarded after centrifuga- treated cells are now permeable to externally added Pronase, tion. The permeable cells were resuspended in the azide DNase-1, deoxy- and ribonucleoside phosphates. The extent buffer. of ATP stimulation in different experiments is 8- to 25-fold, compared to the control. Similar stimulation could be ob- Abbreviations: HH, HL, LL, and DH DNA: respectively heavy served with other ribonucleoside triphosphates, but not by (d = 1.756 g/ml), hybrid (1.729), light (1.703), and denatured monophosphates and only partially by diphosphates. The heavy (1.810) DNA. proportion of viable cells in these experiments was 0.008% or 1296 Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) DNA Synthesis by Detergent-Treated Bacteria 1297

less, and too low to interfere with the assay systems, as verified in reconstruction experiments. Optimal synthesis depends on the presence of cellular DNA, all four deoxyribonucleosides, and ATP; it is sensitive to Pronase and DNase (Table 1). When the time of incubation is increased beyond the 3 hr, the ability of the treated cells to 6 1 ~ dNTP+ATP incorporate dN in the presence of ATP gradually ceases, possibly because of loss of kinases, but the cells can incorporate dN+A/TP dNTP, to an extent that can be increased by mild treatment with DNase-1. This mode of synthesis is not inhibited by N-ethylmaleimide (Table 2) and has been interpreted as a repair process (4). The rate of incorporation is rapid initially, r but decreases on continued incubation at 370C because of nuclease degradation (Fig. lb). With ATP this initial rapid TP increase is inhibited somewhat, but the extent of DNA 0 1 2 3 3 10 20 30 synthesis on prolonged incubation is stimulated anywhere Hours Minute5 from 3.5- to 15-fold (Fig. lb). This process is inhibited by N-ethylmaleimide and has been interpreted as normal DNA FIG. 1. Effect of ATP on incorporation of four deoxynucleo- synthesis (4). Since the amount of repair depends on the sides (dN) and deoxynucleoside triphosphates (dNTP). 3 X 109 of extent of DNA breakage caused in the preparation of per- azide-poisoned cells per ml were incubated at 0C with Brij-58, and aliquots of 0.1 ml were taken at the indicated time and cen- meable cells, one has to take this incorporation into account in trifuged. The sedimented cells were assayed at 370C for 30 min evaluating the extent of stimulation observed with ATP. with dN, dN + ATP, and dNTP. Substrates for DNA synthesis Details concerning the process of this repair synthesis form and cell concentrations were the same as in the legend of Figs. 2 the subject of a subsequent communication. and 3 except that cellular DNA was not labeled. After the reac- In an analysis of the nature of the observed incorporation, tion, the mixture was acid-precipitated and counted for radio- permeable cells were made from cells grown in heavy medium. activity (3). a, Permeable cells obtained as above after 3 hr of in- In certain experiments, these cells were washed and allowed cubation at 0C were used in this assay. b, 0.1 ml of cells were to grow in a light medium for 15 min, so that 10% of the incubated at 370C for different lengths of time with dNTP or DNA molecules were in hybrid form (Fig. 2a). Under these dNTP + ATP and assays were performed as above, with sub- conditions, extensive replication is marked by the production strates as indicated in Fig. 3. of hybrid DNA molecules, as judged by a density shift in a CsCl gradient as well as by the amount of substrate used in hybrid level (Fig. 2b). Under these conditions the template synthesis. On the other hand, repair synthesis should be re- DNA was found to undergo degradation. In these experiments flected by poor incorporation, with no significant change in it is difficult to distinguish repair from normal synthesis. In density profiles. the presence of dNTP, and the absence of ATP, there was Permeable cells, when incubated with dN, and without poor synthesis as reflected by the DNA density and amount of ATP, did not show any incorporation. In the presence of substrate incorporation, and very few light molecules were ATP, a significant proportion of heavy DNA approaches the synthesized (Fig. 3a). With ATP, the resulting profile (Fig. 3b) showed the generation of hybrid and some completely TABLE 1. DNA synthesis (incorporation of deoxynucleosides) light molecules. In addition there were molecules of inter- in permeable cells of B. subtilis mediate density present between hybrid and heavy DNA. The overall distribution of [14C]DNA has moved towards System Activity (%) lighter strata. The incubation with dNTP alone, there was a Complete 100 - ATP 4.8 TABLE 2. DNA synthesis (incorporation of deoxynucleotides) + Pronase, 100/Ag/ml 4.9 in permeable cells of B. subtilis + Bacterial alkaline phosphatase (2 U/ ml)+ATP,2mM 4.6 -any one deoxynucleoside 27.0 System Activity (%) + AMP, 2 mM 4.8 Complete 100 + ADP, 2 mM 28.0 - ATP 31 + ADP, I mM + ATP, 1 mM 64.0 - any one deoxytriphosphate 11 + ADP, 1 mM, + AMP, 1 mM 34.0 - ATP + N-ethylmaleimide, 2 mM (ref. 4) 32 + DNase, 10/Ag/ml 4.4 + heated cells 3.6 + Pronase 1.9 Background 2.2 + DNase 1.6 - Mg++ 3.8 Details of the complete system are given in Fig. 2 and ref. 3. + Pyrophosphate, 25 mM 2.2 0.1-ml aliquots were precipitated with cold trichloroacetic acid onto a Millipore filter soaked in 0.2 M sodium pyrophosphate. Complete system as in Table 1 and Fig. 2. 100% corresponds The precipitate was dried and counted after extensive washing. to 13,000 cpm of 'H. Cellular DNA was not labeled. Substituting 100% represents 8200 cpm of 'H. Cellular DNA was not labeled. NAD or NADP for ATP at similar concentrations did not Azide was present in all the reactions at 0.1 M. stimulate synthesis. Downloaded by guest on September 29, 2021 1298 Biochemistry: A. T. Ganesan Proc. Nat. Acad. Sci. USA 68 (1971)

4- ' b.-16~ U dNTP a I~~f+ATP ~ j.5I12 2 a1 T 2030 40 50~ 0

0od0~~~~~~ 20 50 40 50 5 35 Fractions Fractions FIG. 3. DNA synthesis with deoxynucleoside triphosphates FIG. 2. DNA synthesis with four deoxynucleosides (dN) and and with ATP. Assays as for Fig. 2. Of the four, dATP contained ATP. The reaction mixture, 1 ml, contained 3 X 109 permeable 3H at 30,000 cpm/nmol. Without ATP (a), the total amount of heavy cells, containing 10% of hybrid (HL) DNA molecules incorporation was equivalent to 10 nmol of DNA; with the co- having 45 nmol of [14C]thymidine-labeled DNA (930 cpm/nmol) factor (b), it was 35 nmol. and 45 nmol of each of four deoxynucleosides, of which thymidine was labeled with 3H at 30,000 cpm/nmol. ATP, when used, was at 2 mM. Other reagents were at concentrations described before loss of 33% of the template DNA. The amount of incorpora- (3). After 60 min of reaction at 370C, the isolated DNA contained tion reflected an amount of synthesis equivalent to 20% of 80,000 and 150,000 cpm of 14C and 9H in acid-precipitable form. the template DNA. With ATP and dNTP, there was 7% loss Recovery from the gradient was always more than 80% of the of template DNA and the extent of synthesis here was 3.5 input. 70 fractions were collected from a gradient volume of 8 ml times greater than without ATP. Without ATP, 3.2 and and analyzed as described before (3) for acid-precipitable counts 1Ag DNA was and transforming activity. 1 p&g of standard light B. subtilis DNA with ATP, 11.5 ,g of synthesized. was added to each gradient. a, Profile of a control sample having In order to investigate the nature of the products formed 10% hybrid material. b, Profile of synthesis obtained with dN + in the presence and absence of ATP, I made completely ATP. heavy, permeable cells and performed the above synthesis. The isolated DNA was denatured at alkaline pH and sub- jected to centrifugation in alkaline CsCl. In the presence of TABLE 3. Density distribution of origin, middle, and terminus ATP a significant proportion of light DNA strands, relatively gene activities in DNA after synthesis with dNTP andA TP free of template atoms, was produced (Fig. 4a). There also appear to be heavy chains covalently linked to long stretches Time of synthesis (min) of light atoms and heavy chains linked to short stretches of 0 20 40 light atoms. The former may be the product of semiconserva- tive replication and the latter the products of repair replica- Density position tion. In the control without ATP, a 5-fold lower synthesis was HH HL LL HH HL LL HH HL LL observed as well as loss of template (Fig. 4b). If the synthesis in the presence of ATP represents normal adel6 + 0.94 0.06 0.0 0.84 0.12 0.04 0.44 0.39 0.17 replication, at least in part, the lighter species of molecules leu+ 0.97 0.03 0.0 0.80 0.19 0.01 0.39 0.55 0.07 should be biologically active. Completely heavy SB 168 cells 0.37 0.66 0.03 met5+ 0.96 0.04 0.0 0.75 0.24 0.01 were made permeable and allowed to synthesize DNA for 40 min in the presence of either dN + ATP or of dNTP + HH, HL, and LL refer to heavy, hybrid, and light DNA ATP. The DNA was fractionated in a CsCl gradient and density strata in CsCl gradients. Transformation assays were transformation for that are located at the performed at limiting DNA concentrations, with a multiple assayed by genes and terminus of the chromosome auxotroph (adeI6 leu met5) as recipient bacterium. The values origin, middle, (ade16, leU, represent the proportion of colonies observed at different density and met5) (9). Profiles for adel6+ and mets+ are presented in positions. Fig. 5; all three marker activities and their distributions in Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) DNA Synthesis by Detergent-Treated Bacteria -1299

16

12

8 9 6 'I 4 0) 0 T- x .5 0 ;c I t16 C) a: o12 (-) ._0 O

-0C o 4- 10 20 30 40 50 60 Fractions L 02 10 FIG. 4. Pycnography of the denatured DNA after synthesis with dNTP in the presence (a) and the absence (b) of ATP. Permeable cells made from completely heavy cells were used. The DNA was labeled with [14C] thymine and had a specific activity of 4000 cpm/,gg. Synthesis was performed as for Fig. 3. The product 6 was denatured at pH 12.5, centrifuged in alkaline CsCl at the same pH, and analyzed for 3H and 14C after acid precipitation. 4 the gradient are given in Table 3. In Fig. 5a, the control (incubation without substrates) shows the expected unimodal 2 distribution for two of the genetic activities. With dNTP alone there was no change in the profile of genetic activity. 0 With dN plus ATP (Fig. 5b) more met5+ activity is found near 20 30 40 50 the hybrid position compared to adel6+. The ratio of bio- Fractiona logical activity of molecules with fully heavy density, which FIG. 5. Pycnographic analysis of DNA after synthesis with was originally 3.6 for ade/met, had decreased to 0.9, which dN + ATP and dNTP + ATP. Synthesis was as in Figs. 3b and suggests that under these conditions previously initiated 4b, with completely heavy permeable cells, but for 40 min. DNA chromosomes complete the replication cycle (as judged by from this reaction mixture was centrifuged in CsCl, and 0.01 ml of the shift of met5+ activity to a hybrid level), but that new each fraction was assayed for biological activity (3) by means of initiations are not favored. The amount of synthesis observed competent cells containing mutations for ade16 and met5. Radio- here was always 2 to 8 times less than the one with dNTP activity proffles (not given) were similar to 4b. 100% of adeG + and met5+ represents 2000 and 700 colonies in a, 3500 and 1800 for b, plus ATP. The behavior of other markers close to the terminus and 15,000 and 6300 in c. was similar. The pattern of activity with dNTP + ATP was different. In general, both the ade16+ and mets+ genes have moved to sistent with the interpretation that one is observing both hybrid densities, which suggests not only that certain chromo- initiation and termination of chromosomal replication at somes have finished replication but also that others have least in a large proportion of permeable cells. initiated new rounds of synthesis. There is also adel6+ activity near the light DNA, which does not have a significant amount DISCUSSION of met5+ activity. DNA synthesis, by a mechanism not yet clarified, in this The position occupied by light molecules does not coincide azide-poisoned, pyrophosphate-permeable system is pro- exactly with the position of light standard DNA. I interpret moted several-fold by ATP. Pyridine nucleotides, tested this as the result of a second initiation in a chromosome that because NAD is a cofactor for polynucleotide ligase in B. had the growing point ahead of the adel6+ gene when the cells subtilis (10), do not replace ATP in this reaction. It is possible were made permeable. A kinetic analysis of synthesis at 0, 20, that yet another enzyme activity that is coupled to ATP is and 40 min is presented in Table 3. The distribution pattern involved in DNA synthesis. If replication normally depends of three genes with different chromosomal locations is con- on ATP generation, this would explain the observation that Downloaded by guest on September 29, 2021 1300 Biochemistry: A. T. Ganesan Proc. Nat. Acad. Sci. USA 68 (1971)

in E. coli a cyanide- and CO-sensitive step is required at all was aided by grants from the National Institute of General stages In the presence of these inhibitors Medical Sciences, GM-14108, 2 T01- GM-295 and GB- 8739 of replication (11). from National Science Foundation. The author was a recipient of DNA polymerase and ligases function normally (12),but exten- a U.S. Public Health Service Research Career Program Award, sive synthesis at the growing point is blocked. It was shown GM-50199. earlier (1) that the addition of azide prevents DNA synthesis in B. subtilis. In E. coli, similar inhibitors not only block DNA 1. Ganesan, A. T., and J. Lederberg, Biochem. Biophys. Res. synthesis, but also prevent nucleases from degrading the Commun., 18, 824 (1965). DNA; this suggests that in vitro these systems are energy- 2. Ryter, A., Current Topics in Microbiology and Immunology (Springer-Verlag, New York, 1969), Vol. 49, p. 151. dependent (13, 14). The ATP-stimulated synthesis of DNA is 3. Ganesan, A. T., Proc. Nat. Acad. Sci. USA, 61, 1058 (1968). predominantly dissociated from soluble DNA polymerase 4. Moses, R. E., and C. C. Richardson, Proc. Nat. Acad. Sci. activity, as 95% of the latter is leached out during the process USA, 67, 674 (1970). of making cells permeable. 5. Mordoh, J., Y. Hirota, and F. Jacob, Proc. Nat. Acad. Sci. Synthesis in the absence of ATP results in unimodal dis- USA, 67, 773 (1970). 6. De Lucia, P., and J. Cairns, Nature, 224, 1164 (1969). tribution of several gene activities, consistent with repair 7. Ganesan, A. T., Compt. Rend. Lab. Carlsberg, 31, 149 (1959). replication of template DNA. In contrast, as shown here, 8. Vinograd, J., J. Morris, N. Davidson, and W. F. Dove, Jr., synthesis with ATP results in an asymmetric distribution of Proc. Nat. Acad. Sci. USA, 49, 12 (1963). gene activities that depends on their respective location on 9. Yoshikawa, H., and N. Sueoka, Proc. Nat. Acad. Sci. USA, 49, 559 (1963). the genetic map. Extensive synthesis in the presence of dNTP 10. Laipis, P. J., B. M. Olivera and A. T. Ganesan, Proc. Nat. and ATP indicate both initiation and termination of chromo- Acad. Sci. USA, 62, 289 (1969). some replication. We do not know, however, whether this 11. Denhardt, D. T., and A. B. Burgess, Cold Spring Harbor synthesis reflects the normal in vivo type of chromosome Symp. Quant. Biol., 33, 449 (1968). replication. 12. Cairns, J., and D. T. Denhardt, J. Mol. Biol., 36, 335 (1968). 13. Davern, C., J. Cairns, P. De Lucia, and A. Gunsalus, Sym- The author acknowledges the critical comments and advice of posium on Organizational Biosynthesis (Academic Press, Profs. Joshua Lederberg, Charles Yanofsky, and R. L. Baldwin New York, 1967), Vol. 49. and the expert assistance of Mrs. Rae Ellen Syverson. This work 14. Cairns, J., and C. Davern, J. Mol. Biol., 17, 418 (1966). Downloaded by guest on September 29, 2021