Studies on in Vitro DNA Synthesis:* Purification of Dna C Gene Product

Proc Nat. Acad. Sci. USA Vol. 70, No. 8, pp. 2369-2373, August 1973

Studies on In Vitro DNA Synthesis:* Purification of dna C Gene Product Containing dna D Activity from Escherichia coli (OX174 DNA/DNA replication/DNA initiation) SUE WICKNER, IRA BERKOWER, MICHEL WRIGHT, AND JERARD HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461 Communicated by Harry Eagle, May 10, 1973

ABSTRACT The conversion of 45X174 single-stranded Preparation of Receptor Crude Extracts and Receptor Am- DNA to duplex DNA by extracts of E. coli requires products monium Sulfate Fractions. Receptor crude extracts were pre- of the E. coli DNA replication genes. By use of this complementation system, the dna C gene product has been puri- pared as described (12). Receptor ammonium sulfate fractions fied from wild-type E. coli as well as from a dna C tempera- were prepared as follows: cells, grown and frozen (16), were ture-sensitive mutant. The latter preparations are temper- thawed in an ice-water bath. They were lysed with 0.2 mg/ml ature sensitive when compared to the wild-type gene of lysozyme, 50 mM Tris * HOl (pH 8.5), 1 mM dithiothreitol, product. The dna C and dna D gene products copurify, have similar characteristics, are both temperature sensitive in 20 mM EDTA, 0.1% Brij 58, and 150 mM KCI for 20 min at preparations from dna C temperature-sensitive cells, and 00 and centrifuged for 40 min at 50,000 X g at 4°. The super- are both undetectable in preparations from dna D tem- natant was adjusted to a final concentration of 4% with a perature-sensitive cells. solution of 20% streptomycin sulfate and centrifuged at Escherichia coli (ts) 10,000 X g for 5 min. The supernatant was adjusted to 40%Q temperature-sensitive mutants, which fail saturation with sulfate and to replicate their chromosome at elevated temperatures, have saturated, neutralized ammonium in in centrifuged at 10,000 X g for 10 min. The pellet was suspended been placed seven loci six regions on the E. coli chromo- in of 10 mM Tris-HCl some (1-8). The dna E gene product has been identified as 0.1 volume (of original crude extract) DNA polymerase III, the DNA polymerase directly involved (pH 7.5), 1 mM dithiothreitol, 0.5 mM EDTA, and 15% in DNA replication (9, 10), while the dna F gene product has glycerol and dialyzed against the same buffer for 2 hr. After been characterized as ribonucleotide reductase (11). The dna G dialysis the salt concentration was 0.1 M. The receptor ammonium sulfate fraction was then frozen in small gene product has been extensively purified but its enzymatic portions. function in DNA synthesis is unknown (12, 13). Extracts of E. coli catalyze dNMP incorporation dependent TABLE 1. Temperature sensitivity of qX174 DNA-dependent upon OX174 DNA (14). The reaction has been reported to de- DNA synthesis in ammonium sulfatefractionsfrom dna C ts cells pend on E. coli gene products dna A and B (15) as well as on dna C, D, E, and G (16). Stimulation of inactive crude extracts Temperature of prepared from ts cells by fractions from wild-type cells has incubation provided a complementation assay for purification of these dna gene products (12, 15, 16). This communication reports Min of 200 350 the isolation of the dna C gene product and the relationship E. coli strain used for incuba- (dTMP incorporated, between dna C and dna D gene products. Genetic evidence ammonium sulfate fraction tion pmol) that dna C and dna D are one gene has been reported by HMS-83 (wild type) 15 8.7 10.7 Wechsler, using in vivo complementation tests (17). 60 29.0 35.3 CT28-3b (dna C ts) 20 5.4 0.2 MATERIALS AND METHODS 40 12.5 0.2 Unless otherwise specified, materials and reagents were as PC22 (dnaC ts) 20 1.6 0.3 described (12). 40 3.4 0.5 60 4.9 0.7 Bacterial Strains. The following E. coli strains were used: BW824 (dna C ts) 20 30.4 3.6 HMS-83 (pol Al, pol B1, thy, lys) (18); PC22 (pol Al, his, strr, PC1 (dna C ts) 30 <0.5 - arg, mtl, dna C2 ts) (9); PC79 (pol Al, his strr, mtl, dna D7 ts) PC79 (dna D ts) 30 <0. 5 (9); PC1 (leu, thy. strr, dna Cl) (4); BW824, (thy A, dra, leu, strr, ampr, dna 325 ts) (6); CT28-3b(his, pro, arg, thy, strr, dTMP incorporation by ammonium sulfate fractions was thr, leu, dna C ts) (7). measured with buffer, dNTP, ATP, jX174 DNA, and spermidine additions as described for the complementation assay. The time and temperature of incubation were as indicated. Protein addi- Abbreviation: ts, temperature sensitive. tions were as follows: 0.2 mg of the HMS-83 preparation, 0.34 mg * This is paper IV in a series. Papers I, II, and III are refs. 16, 22, of the CT28-3b, 0.4 mg of the BW824, 0.4 mg of the PC22, 0.9 mg and 12, respectively. of the PC1, and 0.8 mg of PC79. 2369 Downloaded by guest on September 30, 2021 2370 Biochemistry: Wickner et al. Proc. Nat. Acad. Sci. USA 70 (1978) from dna C ts strain PC1 or dna D ts strain PC79. In contrast, 12 the rate of synthesis at 350 by fractions from wild-type cells was nearly the same as the rate at 20°. Thus the dna C gene

0 10 product is involved in the OXi74 DNA-dependent system. E When equal mixtures of ts and wild-type preparations were 8 incubated together at 200 or 350, dTMP incorporation was 2 more than additive, suggesting that wild-type fractions con-

0 tained an excess of dna C gene product. 0 6 0 The temperature inactivation of ammonium sulfate prep- 4 arations of CT28-3b was reversible (Fig. 1). During an incubation of 45 min at 350 there was no detectable dTMP incorporation. When the temperature was lowered to 20° after 2 15 min at 350, dTMP incorporation occurred and increased linearly for at least 30 min. This result is consistent with the 5 15 25 35 45 demonstration in vivo that the temperature-sensitive defect Minutes in initiation of DNA replication in this mutant is reversible FIG. 1. Reversible temperature sensitivity of OX174 DNA- (7). dependent DNA synthesis by preparations from dna C ts cells. dTMP incorporation catalyzed by ammonium sulfate fractions Purification of dna C Gene Product. The purification, yield, from E. coli CT28-3b was measured as described in Table 1 with and ratio of dna D activity to dna C activity are summarized 0.34 mg of protein. Incubation at 200, 0; incubation at 350, A; in Table 2. The crude extract could not be assayed for dna C incubation for 15 min at 35° followed by shift to 200, 5. and dna D activities because dTMP incorporation was independent of receptor crude extract and receptor ammonium sulfate fraction. The extent of purification from the ammo- Both receptor crude extracts and receptor ammonium sulfate nium sulfate fraction through the glycerol gradient step was fractions were thawed immediately before use. about 175-fold, with a 20% yield. The dna C and dna D complementing activities were not separated by the purification Complementation Assay for dna C and dna D. Each assay procedure (Table 2). Furthermore, all fractions that contained (0.05 ml) contained 20 mM Tris HCl (pH 7.5), 10 mM MgCl2, one activity contained both. As shown below, stimulation of 4 mM dithiothreitol, 0.04 mM each of dATP, dGTP, dCTP, the dna D complementation assay is due to the dna C gene and [a-32P]dTTP (200-500 cpm/pmol), 10ug/ml of rifampicin, product. 5 mM ATP, 2.5 mM spermidine .11Cl, 500 pmol of 4X174 DNA, 0.01 ml (15 mg/ml of protein) of receptor crude extract Properties of dna C and dna D Complementation Assays. prepared from E. coli strain PC22 or PC79, 0.005 ml (50 mg/ These complementation assays required OX174 DNA, ATP, ml of protein) of receptor ammonium sulfate fraction prepared Mg+2, -receptor crude extract, receptor ammonium sulfate from the same E. coli strain as the receptor crude extract, and fraction, and purified dna C gene product (Table 3). The re- protein fractions as indicated. After incubation at 250 for 20 quirement for both receptor ammonium sulfate fraction and min, the reaction was stopped and the sample was precipitated receptor crude extract was not absolute; large amounts of with acid as described (19). One unit (U) of dna C activity in- ammonium sulfate fraction would substitute for the crude corporated 1 nmol of dTMP in 20 min at 250 under the condi- extract requirement and, similarly, large amounts of crude ex- tions described above, using receptor crude extract and recep- tract would substitute for the ammonium sulfate fraction. tor ammonium sulfate fraction prepared from E. coli strain However, the combination, as described in Methods, gave the PC22. dna D activity is defined as above, using receptor crude most reproducible results. As shown, addition of spermidine extractand receptorammonium sulfate fraction prepared from stimulated the reaction but was not always an absolute re- E. coli strain PC79. Assay of complementingactivityof purified quirement. dna C gene product varied with the preparation of receptor Under the conditions of the assay, dTMP incorporation was crude extract and receptor ammonium sulfate fraction used. dependent on the amount of purified dna C gene product Specific activity refers to units of activity per mg of protein; added. For example, with 0.2, 0.4, or 1.0 Mg of DEAE- protein was measured by the method of Bucher (20). cellulose fraction, 4.0, 8.1, and 24.8 pmol of dTMP were incorporated, respectively, with assay conditions for dna C RESULTS complementation; with the same amounts of protein, 1.0, 3.0, Involvement of dna C Gene Product in X174 DNA- and 13.3 pmol of dTMP were incorporated, respectively, with Dependent dNMP Incorporation. While our previous studies the dna D complementation assay. Incorporation of dTMP (16) showed that OX174 DNA-dependent activity was not increased with time for at least 40 min at 250; with 0.4 jsg of detectable in crude extracts of E. coli dna C ts cells, we have DEAE-cellulose fraction after 10, 20, 30, and 40 min of incuba- now been able to observe 4X174 DNA-dependent activity. tion, 1.3, 8.1, 14.8, and 25.5 pmol of dTMP were incorporated, Ammonium sulfate fractions prepared from E. coli dna C ts respectively, with the dna C complementation assay while strains PC22, BW824, CT28-3b, and PC1, dna D ts strain 0.3, 3.0, 5.6, and 8.1 pmol of dTMP were incorporated, re- PC79, and wild-type strain HMS-83 were assayed for 4X174 spectively, with the dna D complementation assay at the DNA-dependent dTMP incorporation at 20° and 35°. The same times. A stimulation of activity (as much as 10-fold) rate of synthesis at 350 by fractions from the ts strains was was observed when the assay mixture for dna C or dna D com- much reduced from the rate at 200 (Table 1). There was no plementation was previously incubated for 5-30 min at 250 detectable dTMP incorporation observed in preparations in the absence of dNTPs, 4X174 DNA, and spermidine and Downloaded by guest on September 30, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) dna C Gene Production of Escherichia coli 2371

then incubated for 20 min at 250 after addition of these com- TABLE 3. Requirements for dna C and D ponents. complementation assays The acid-insoluble product labeled with dTMP formed after a 10- or 20-min incubation in the dna C or dna D comple- dna C dna D mentation reaction sedimented in neutral sucrose gradients at comple- comple- the position of RFJI. In alkaline sucrose gradients, of the menta- menta- 75% tion tion (dTMP incorpor- TABLE 2. Purification of dna C gene product Additions ated, pmol) Complete 6.1 7.9 Ratio of Omit rifampicin 5.8 9.1 Specific dna C Omit spermidine 5.3 0.7 activity % to dna D Omit Mg+, 0.3 <0.2 Fraction Total U U/mg Recovery activity Omit ATP <0.2 0.2 High-speed Omit OX174 DNA <0.2 <0.2 supernatant - - - Omit dna C or D receptor crude <0.2 2.4 Ammonium sulfate Omit dna C or D receptor ammonium fraction (0-40%) 225 0.4 100 1.1 sulfate fraction <0.2 0.4 DNA-agarose Omit dna C gene product 0.6 0.9 eluate 160 2.5 71 1.3 DEAE-cellulose The assay conditions were as described in Methods, with 0.006 eluate 70 27 31 1.6 U of dna C activity (DEAE-cellulose fraction). Glycerol gradient* fraction 45 70 20 1.4 product sedimented at the position of full-length linear strands. * This step was performed with a small portion of the DEAE- Properties of dna C Gene Product. Purified dna C gene cellulose eluate. The results presented are calculated for the use product possesses a molecular weight of about 25,000. We de- of the entire DEAE-cellulose eluate fraction. this value sedimenting the DEAE-cellulose frac- All purification steps were done at 4°. An ammonium sulfate termined by fraction, prepared from 100 g of E. coli HMS 83 as for preparation tion through a glycerol gradient using the conditions de- of receptor ammonium sulfate fractions, was dialyzed against scribed above with E. coli alkaline phosphatase and horse- 500 ml of 0.01 M potassium phosphate buffer (pH 7.5), 1 mM radish peroxidase as markers. As shown in Fig. 2, dna C and dithiothreitol, 0.5 mM EDTA, and 20% glycerol (changed 4 dna D complementing activities sedimented coincidentally. times) over 3 hr. DNA-agarose column chromatography: The dna C activity was sensitive to N-ethylmaleimide. The dialyzed ammonium sulfate fraction was diluted to 225 ml with DEAE-cellulose fraction was incubated with 10 mM N-ethyl- 2 mM potassium phosphate buffer (pH 6.8), 1 mM dithiothreitol, maleimide 10 min at 250. The mixture was then adjusted to 0.5 mM EDTA, and 10% glycerol (Buffer A). The final salt 50 mM with dithiothreitol and assayed for dna C and dna D concentration was 5 mM. The sample was applied to a 100-ml complementing activities. Both activities were completely column of denatured calf-thymus DNA-agarose (21) that was inhibited. equilibrated with Buffer A. The column was washed with 50 ml of in- Buffer A, and dna C activity was eluted with 50 mM Tris HCl dna C and dna D complementing activities were heat (pH 7.5), 20% glycerol, 1 mM dithiothreitol, 0.5 mM EDTA activated at similar rates. The DEAE-cellulose fraction was (Buffer B) containing 1 M NaCl. The 1 M salt eluate was ad- heated for various times at 450 and then assayed at 250 under justed to 50% saturation with solid ammonium sulfate (29.1 g/100 the usual conditions. Both activities were 50% inactivated ml). The precipitate was collected by centrifugation, dissolved in after 3-4 min at 450. 5 ml of Buffer C, and dialyzed against 1 liter of the same buffer for dna C and (Ina D activities bind weakly to DNA. They did 3 hr. DEAE-cellulose column chromatography: The DNA-agarose not bind to DNA-agarose at pH 7.5 or higher, and when bound fraction (5 ml) was diluted to 10 ml with Buffer B and applied to a 35-ml DEAE-cellulose column equilibrated with Buffer B. The column was washed with 50 ml of Buffer B, and 5-ml fractions were collected; dna C activity appeared in the effluent, which 2 8.0 -4.040° passed directly through the column. Active fractions were pooled E 0 (15 ml) and adjusted to 60% saturation with solid ammonium sulfate (36.1 g/100 ml); the pellet was collected by centrifugation. 66.0 - 310 'CD CD This material was dissolved in 1 ml of Buffer B. The activity at 0 . - 80 2.0 1 this stage was unstable if stored at protein concentrations lower C than 0.5 mg/ml. At a protein concentration of 2 mg/ml or higher, E30 : dna C activity has been stable for 2 months, even with repeated I. 2.0 1.0 freezing (-10°) and thawing. Glycerol gradient centrifugation: E A portion of the DEAE-cellulose fraction was dialyzed for 30 min against 0.2 M KCl, 0.02 M potassium phosphate buffer (pH 7.5), o ~~4 8 12 16 20 24 28 2 mM dithiothreitol, and 0.5 mM EDTA. The dialyzed sample Fraction Number 0 (0.2 ml) was layered on a 5-ml linear gradient of 10-30% glycerol FIG. 2. Glycerol gradient centrifugation of dna C gene prod- in the same buffer and centrifuged in a Spinco SW50.1 rotor at uct. Glycerol gradient centrifugation was performed with the 50,000 rpm for 30 hr. The dna C activity sedimented through half DEAE-cellulose fraction as described for purification of dna C. the gradient. This fraction was unstable possibly due to its low Fractions were assayed for dna C activity (-) and dna D activity protein concentration. (0). Downloaded by guest on September 30, 2021 2372 Biochemistry: Wickner et al. Proc. Nat. Acad. Sci. USA 70 (1978) TABLE 4. Measurement of dna gene products in 12 purified preparations 10 Purified E. coli dna dna gene product measured 8 gene product added B C D E G 4) 6 (pmol of dTMP incorporated) 2 4 B 40 <0.5 <0.5 <0.5 <0.5 a, C (D) <0.5 57 33 <0.5 <0.5 a0 2 E (DNA polymerase III) <0.5 <0.5 <0.5 39 <0.5 - G <0.5 <0.5 <0.5 <0.5 42 0

C a.~0 dna gene products were measured as follows: dna E (DNA 8 polymerase III) was assayed with DNase-treated salmon-sperm DNA (19); dna B and dna G gene products were assayed with the 6 iX174 DNA complementation system (12); dna C and dna D 4 assays were performed as described in Method. Additions were 30 ng of dna B, 2.0 ug of dna C, 0.1 ug of DNA polymerase III, 2 and 0.5 jg of dna G.

20 40 60 MInutes DNase-treated DNA as primer-template). Purified dna G FIG. 3. Temperature sensitivity of dna D complementing gene product and purified DNA polymerase III specifically activity isolated from dna C ts and wild-type cells. 0.11 mg of complemented dna G ts and dna E ts receptor crude extracts, protein of DEAE-cellulose fraction from dna C ts cells (CT28-3b) respectively (6). Similarly, purified dna B gene product (puri- and 0.02 mg from wild-type cells (HMS-83) were assayed for dna fication unpublished) specifically complemented dna B ts D complementing activity for various times at the temperature receptor crude extracts. In these experiments, the dna A gene indicated. Wild-type dna C gene product (A); ts dna C gene product was not measured since we have been unable to con- product (0); mixture of 1/2 wild-type and 1/2 ts dna C gene struct a 4X174 DNA-dependent complementation assay that products (X); receptor crude extract plus receptor ammonium requires the dna A gene product. t sulfate fraction minus purified dna C gene product (0); wild-type or ts dna C gene product minus receptor crude extract and re- Purification of Thermolabile dna C Gene Product from dna C ceptor ammonium sulfate, (0). ts Cells. dna C activity was purified as described above through the streptomycin sulfate and 0-40% ammonium sulfate steps from wild-type (HMS-83) and dna C ts (CT28-3b) at pH 6.8, they were eluted with 0.15 M NaCl. Preincubation cells. The fractions, after dialysis, were applied to DEAE- of the glycerol gradient fraction with 4C-labeled 4X174 or cellulose columns as described for purification of dna C ac- fd DNA did not protect these DNAs from degradation by tivity; dna C complementing activity passed through the nuclease SI or Neurospora nuclease, under conditions in which columns. The activity from the dna C ts strain was unstable; E. coli unwinding protein rendered the DNAs resistant to these all activity was lost after 6 hr on ice. Both dna C and dna D nucleases by binding tightly to the DNA (22, 23). complementing activities were temperature sensitive at 350 in Several enzymatic activities were measured in the DEAE- the DEAE-cellulose fraction prepared from the ts mutant cellulose fraction. This fraction did not catalyze incorporation when compared to the DEAE-cellulose fraction isolated from of rNMPs with OX174, fd, or calf-thymus DNA as template, the wild type. Fig. 3 shows the rate of synthesis in the dna D or incorporation of dNMPs with DNase-treated salmon- complementation assay at 20° and 35°. Since the preparation sperm DNA (19) or fd DNA RNA hybrids as primer tem- purified from dna C ts cells was ts in the dna C complementa- plates. It was free of detectable ATP-dependent and -indepen- tion assay, the material obtained with this assay and purifica- dent DNase activity (measured by degradation of "4C-labeled tion procedure contains the dna C gene product. Since the ac- OX174, fd, and denatured T7 DNA to acid-soluble material). tivity purified from the dna C ts cells was also ts in the dna D The DEAE-cellulose fraction was also free of detectable complementation assay, either the product of the dna C gene DNA-dependent and -independent ATPase activity. The is also the product of the dna D gene (i.e., dna C and dna D glycerol gradient fraction contained no detectable RNase are one locus) or inactivation of the dna C gene product re- activity (measured by degradation of ['H]poly(A) to acid- sults in inactivation of the dna D gene product. soluble material). This fraction contained detectable RNase H Receptor ammonium sulfate fractions isolated from dna D activity (1.1 psg of protein degraded 11.2 pmol of ['H]poly(A) ts cells (PC79) lacked dNMP-incorporating activity depen- to acid-soluble material in the presence of poly(dT) in 30 min dent upon OX174 DNA. Furthermore, these fractions were at 380). inactive in both dna D and dna C complementation assays. dna C gene product, purified through the DEAE-cellulose The lack of dna D complementing activity in fractions pre- step, stimulated 4X174 DNA-dependent dNMP incorpora- pared from the dna D ts mutant is probably due to instability tion only in dna C and dna D complementation assays (Table of the dnaD ts gene product. The lack of dnaC complementing 4). It was free of dna B and dna G gene products [measured in similar complementation assays with inactive receptor ex- t The dna A ts strains that have been studied include CRT4638 tracts prepared from dna B and dna G ts cells (6)] and dna E (pol A, strain obtained from Dr. Y. Hirota), CRT837 (1), PC5 gene product (measured as DNA polymerase activity with (4), E177 (2), and E508 (2). Downloaded by guest on September 30, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) dna C Gene Production of Escherichia coli 2373

activity in the dna D ts preparation again suggests either that system, it has been possible to purify the products of E. coli the products of the two genes are the same or that inactiva- dna B, C, E, and G genes. OX174 DNA-dependent dNMP in- tion of one of the gene products results in inactivation of the corporation is not observed when these components are added other. together with or without DNA polymerase stimulatory proteins (22), but is observed when a crude fraction from E. DISCUSSION coli is added. Thus, this provides an assay for other E. coli The use of the soluble 4X174 DNA-dependent dNMP in- proteins involved in this system and possibly involved in E. corporating system requiring the dna gene products of E. coli coli replication. has facilitated isolation of these proteins. The dna B, C, E, We thank Drs. J. Wechsler, B. Wolf, P. Carl, and Y. Hirota for and G gene products have been purified with this system; the their generosity in supplying bacterial strains. This work was present report concerns isolation of the dna C gene product. supported by grants from the National Institutes of Health Involvement of the dna C gene product in the 4X174 DNA- (GM-13344) and the American Cancer Society (NP-890D). S.W. system is demonstrated the sensi- is a trainee of the National Institutes of Health, I.B. is a Medical dependent by temperature Scientist Trainee, and M.W. is a fellow of the Jane Coffin Childs tivity of this activity in extract of a dna C ts mutant when Memorial Fund for Medical Research. Schubach et al. have compared to wild-type preparations. (7) 1. Hirota, Y., Ryter, A. & Jacob, F. (1968) Cold Spring Harbor shown that the ts defect in DNA synthesis by this mutant is Symp. Quant. Biol. 33, 677-693. reversible in vivo. Similarly, it is shown here that the tempera- 2. Wechsler, J. A. & Gross, J. D. (1971) Mol. Gen. Genet. 113, ture sensitivity of the in vitro OX174 DNA-dependent system 273-284. in preparations of this dna C ts mutant is reversible. 3. Hirota, Y., Mordoh, J. & Jacob, F. (1970) J. Mol. Biol. 53, 369-387. The assay procedure used to purify the dna C gene product 4. Carl, P. L. (1970) Mol. Gen. Genet. 109, 107-122. appears valid since the purification procedure yielded a ts 5. Marinus, M. G. & Adelberg, E. A. (1970) J. Bacteriol. 104, protein from a dna C ts mutant that was also ts in the dna D 1266-1272. complementation assay. While the dna C and dna D genes 6. Wolf, B. (1972) Genetics 72, 569-593. map on E. coli were 7. Schubach, W. H., Whitmer, J. D. & Davern, C. I. (1973) close together the chromosome (2, 4), they J. Mol. Biol. 74,205-221. originally thought to be different loci. This conclusion was 8. Wechsler, J. A., NOsslein, V., Otto, B., Klein, A., Bonhoeffer, based on the observations that dna C ts mutants can support F., Herrmann, R., Gloger, K. & Schaller, H. (1973) J. phage X synthesis at the nonpermissive temperature, while Bacteriol. 113, 1381-1388. dna D ts mutants cannot, and that dna C ts mutants complete 9. Gefter, M. L., Hirota, Y., Kornberg, T., Wechsler, J. & Barnoux, C. (1971) Proc. Nat. Acad. Sci. USA 68, 3150-3ti3. rounds of chromosome replication at the elevated tempera- 10. Ntisslein, V., Otto, B., Bonhoeffer, F. & Schaller, H. (1971) ture but fail to start new rounds, while dna D ts mutants stop Nature New Biol. 234, 285-286. DNA synthesis immediately when shifted to the nonpermis- 11. Fuchs, J. A., Karlstom, H. O., Warner, H. R. & Reichard, P. sive temperature (4). Evidence against dna C and dna D being (1972) Nature New Biol. 238, 69-71. 12. Wickner, S., Wright, M. & Hurwitz, J. (1973) Proc. Nat. separate loci has been presented by Wechsler (17). He has Acad. Sci. USA, 70, 2969-23M8 been unable to show in vivo complementation between dna C 13. Nusslein, V., Bonhoeffer, F., Klein, A. & Otto, B. (1972) in and dna D ts mutants, suggesting that the dna C and dna D The Second Annual Harry Steenbock Symposium, eds. Wells, loci may be identical. Our data support this suggestion but do R. & Inman, R. (University Park Press, Maryland), in press. not eliminate other possibilities, including inactivation of the 14. Wickner, W. T., Brutlag, D., Schekman, R. & Kornberg, A. (1972) Proc. Nat. Acad. Sci. USA 69, 965-969. dna C gene product resulting in inactivation of the dna D 15. Schekman, R., Wickner, W. T., Westergaard, O., Brutlag, gene product. D., Geider, K., Bertch, L. L. & Kornberg, A. (1972) Proc. The enzymatic activity that corresponds to the dna C gene Nat. Acad. Sci. USA 69, 2691-2695. product is unknown. The purified preparation did not catalyze 16. Wickner, R. B., Wright, M., Wickner, S. & Hurwitz, J. incorporation of ribo- or deoxyribonucleotides. It was free of (1972) Proc. Nat. Acad. Sci. USA 69, 3233-3237. 17. Wechsler, J. A. (1972) in The Second Annual Harry Steenbock DNA-dependent and -independent ATPase activities and Symposium, eds. Wells, R. & Inman, R. (University Park ATP-dependent and -independent DNase and RNase. Al- Press, Maryland), in press. though mutations in dna C affect initiation of chromosome 18. Campbell, J. L., Soll, L. & Richardson, C. C. (1972) Proc. Nat. replication but not chain elongation in vivo (4, 6, 7), incuba- Acad. Sci. USA 69, 2090-2094. 19. Wickner, R. B., Ginsberg, B., Berkower, I. & Hurwitz, J. tion of an ammonium sulfate fraction from a ts dna C mutant (1972) J. Biol. Chem. 247, 489-497. with 4X174 DNA and ATP (but no dNTPs) at low temper- 20. Bucher, T. (1947) Biochim. Biophys. Acta 1, 292-314. ature followed by a shift to nonpermissive temperature did 21. Schaller, H., Nusslein, C., Bonhoeffer, F. J., Kurz, C. & not lead to dNTP incorporation upon addition of dNTPs (un- Neitzschmann, I. (1972) Eur. J. Biochem. 26, 474-481. 22. Hurwitz, J., Wickner, S. & Wright, M. (1973) Biochem. published results). Thus, if a complex were formed, it does Biophys. Res. Commun. 51, 257-267. not survive at the nonpermissive temperature. 23. Sigal, N., Delius, H., Kornberg, T., Gefter, M. L. & Alberts, By use of complementation in the 4X174 DNA-dependent B. (1972) Proc. Nat. Acad. Sci. USA 69, 3537-3541. Downloaded by guest on September 30, 2021