Proc. Nat. Acad. Sci. USA Vol. 72, No. 3, pp. 921-925, March 1975

Interaction of Escherichia coli dnaB and dnaC(D) Products In Vitro (DNA replication/DNA-stimulated ATPase) SUE WICKNER* AND JERARD HURWITZt * Viral Carcinogenesis Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 20014; and t Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York, 10461 Contributed by Jerard Hurwitz, December 24, 1974

ABSTRACT Purified E. coli dnaB and dnaC(D) gene tation in the 4X174 DNA-synthesizing system. The assay products interact physically and functionally in vitro. This interaction was demonstrated as follows: (a) A com- conditions, definitions of units, and purification procedures plex of dnaB and dnaC(D) gene products was isolated by were described previously for dnaB (19, 21) and dnaC(D) gel filtration; ATP specifically was required for isolation gene products (19, 16). The dnaB gene product contained no of the complex. (b) The DNA-independent ribonucleoside detectable dnaC(D) complementing activity (<5 X 10-4 triphosphatase activity associated with dnaB gene product U/0.4 U of dnaB), and dnaC(D) gene product contained no was inhibited by dnaC(D) gene product. (c) The dnaC(D) gene product was protected from inactivation by N-ethyl- detectable dnaB activity [<5 X 10-4 U/0.5 U of dnaC(D)]. maleimide by the combination of dnaB gene product and The addition of dnaB (0.12 U) to the dnaC(D) comp)lementa- ATP; this protection required ATP specifically. tion assay system with dnaC(D) (0.015 U) did not affect the dnaC(D) complementing activity; the addition of dnaC(D) Some of the proteins involved in Escherichia coli DNA rep- (0.15 U) to the dnaB complementation assay system with lication have been defined by temperature-sensitive mutants dnaB (0.012 U) did not affect the dnaB complementing ac- that do not replicate their DNA at elevated temperatures. tivity. The that determine these proteins have been designated dnaA (1), dnaB (2), dnaC(D) (3), dnaE (4), dnaG (3), dnafH Assay for A TPase. Reaction mixtures (0.03 ml) contained (5), dnaH (6), dnaI (7), dnaP (8), lig (9), and polA (10). 50 mM Tris HClI, pH 7.5, 1 mM dithiothreitol, 0.4 mM Mutants in dnaE contain thermolabile DNA III MgCl2, 0.05 mg/ml of bovine serum albumin, 0.2 mM (11, 12); lig mutants contain thermolabile DNA ligase (9, 13, ['y-32P]ATP (50 cpm/pmol), and protein fractions as in- 14); and polA mutants are temperature-sensitive for growth dicated. After 30 min at 30°, Pi was determined by the method when they contain thermolabile 5' to 3' exonuclease (exo- of Conway and Lipmann (22). One unit (U) of ATPase nuclease VI) (15). Of the other proteins known to be involved catalyzed the production of 1 ,umol of 32Pi using the above in E. coli DNA replication, dnaB, C(D), and G have been iso- conditions. lated using in vitro complementation assays in which protein RESULTS preparations from wild-type cells stimulate 4X174 DNA- dependent DNA synthesis in heat-inactivated crude extracts Physical Association of dnaB and dnaC(D) Gene Products. of dna temperature-sensitive cells (16-19). Each of these dna The dnaC(D) gene product was detected (as measured by gene products has been isolated from both wild-type and stimulation in the 4X174 DNA-dependent complementation temperature-sensitive strains and the thermolability of the assay) physically associated with the dnaB gene product (also temperature-sensitive protein has been demonstrated in the measured by the OX174 DNA-dependent complementation 4X174 DNA-dependent complementation system. As yet, no system) when the two purified proteins were mixed and sub- enzymatic activity has been found associated with dnaG or jected to gel filtration through BioGel A-5m agarose in the dnaC(D) gene products. The dnaC and dnaD gene products presence of ATP (Fig. 1A). Incubation of dnaC(D) with dnaB have been shown to be identical in vitro (16) and in vivo (20); gene product prior to gel filtration was not essential and did the protein will be referred to here as dnaC(D) gene product. not result in an increased amount of dnaC(D) associated with Purified dnaB gene product contains ribonucleoside triphos- dnaB. Fig. 1B shows that in the presence of ATP and in the phatase activity that is stimulated by single-stranded DNA. absence of dnaB gene product, dnaC(D) eluted as expected These triphosphatase activities and dnaB complementing for a protein with a molecular weight of about 25,000 (16). activity copurify over the last 20-fold of a 40,000-fold puri- Fig. 1C shows that dnaB gene product in the presence of fication procedure (21). The purpose of this report is to de- ATP and absence of dnaC(D) gene product eluted as expected scribe the physical and functional interaction of two of these for a protein with a molecular weight of about 250,000 (18) $. proteins involved in replication, dnaB and dnaC(D) gene pro- It eluted broadly, suggesting, as shown before (21), that dnaB ducts. gene product can exist in multiple forms that are active in the MATERIALS AND METHODS t Similar results to those shown in Fig. lA and B were obtained Materials, Reagents, and Methods, unless otherwise indi- when columns were developed with 50 mM Tris-HCl (pH 7.5) cated, were as described (16-19). and 50 mM KCl in place of 30 mM potassium phosphate (pH 6.0). However, at pH 7.5, dnaB complementing activity could Isolation of dnaB and dnaC(D) Gene Products. dnaB and not be recovered in the absence of ATP in the presence or absence dnaC(D) gene products were assayed by in vitro complemen- of dnaC(D) gene product. 921 Downloaded by guest on October 1, 2021 922 Biochemistry: Wickner and Hurwitz Proc. Nat. Acad. Sci. USA 72 (1975)

DNA-dependent complementation assay. Since the A. dnaB + dnaC gene products + ATP Included OX174 gene product was not significantly 0.16 void volume voume 0.16 elution profile of dnaB affected by its interaction with dnaC(D) gene product, proba- 0.08 0.08 bly only one or a few dnaC(D) molecules are associated with one native dnaB molecule. were the B. dnaC gene product + ATP When dnaB and dnaC(D) gene products mixed in 0.24 absence of ATP and filtered through a column in the absence of ATP, the two proteins were not associated with each other 0.16 (Fig. 1D). Furthermore, most of the dnaB activity was ex- cluded from the column; the nature of this aggregation has 0.08 not been studied, nor has it been demonstrated that it was catalyzed exclusively by the dnaC(D) gene product. In the 0.16 C. dnaB gene product + ATP absence of ATP, dnaB and dnaC(D) gene products alone eluted as they had in the presence of ATP (results similar to 0.08 those shown in Fig. 1B and C). Fig. 1E shows that dnaB and dnaC(D) activities were not associated with each other if they cl D. dnaB + dnaC gene products-ATP were incubated together for 30 min at 300 in the presence of E 024 0.24 ATP and then filtered through the agarose column in the 0.16 0.16 absence of ATP. Thus, ATP is required for detecting the physical complex of dnaB and dnaC(D) gene products. 0.08 0.08 Under conditions as shown in Fig. 1A but with 0, 1, 5, 50, and 11 500 AM ATP, 0, 17, 33, 48, and 52% of the dnaC(D) activity, E. dnaB + dnaC gene products incubated+ATP, respectively, was associated with the dnaB gene product. gel filtration-ATP The nucleotide requirement was specific for ATP. Under the conditions described in Fig. 1A, no dnaC(D) activity was 024 detected associated with dnaB gene product when ATP was 0.16 0.16 replaced with 10AM dATP, dGTP, dTTP, UTP, CTP, GTP, Bry-methylene-ATP, or ADP (<5% of that associated with 0.08 0.08 dnaB gene product in the presence of ATP). The fate of ATP in this reaction is unknown. No detectable 91 _ 0 AMP was produced by dnaB, dnaC(D), or the combination 024 F. 1X dnaB + '/2X dnaC + ATP of gene products. No detectable ['y-'2P]ATP, [a-'2P]ATP, or 0.16 0.16 [3H]ATP was bound by dnaB, dnaC(D), or the combination, as measured by Millipore binding, but more sensitive means 0.08 0.08 of measuring weak binding have not been used as yet. At- , -4.-i I tempts to determine if a small amount of ATP were hy- 4 8 12 16 20 24 28 32 drolyzed by the interaction of dnaB with dnaC(D) gene prod- Fraction Number uct were complicated by the ribonucleoside triphosphatase activity associated with dnaB (21), which, as shown below, agarose of FIG. 1. BioGel A-5m gel filtration dnaB, dnaC(D), dnaC(D) gene product inhibits. and the combination of gene products. (A) A reaction mixture The interaction of dnaB and dnaC(D) gene products de- (0.075 ml) containing dnaB (1.2 U), dnaC(D) (1.5 U), 0.5 mM pended on the ratio of these two proteins. In the experiment ATP, 1.0 mM MgCl2, 0.05 mg/ml of bovine serum albumin, 30 mM potassium phosphate (pH 6.0), and 10 mM dithiothreitol shown in Fig. 1A, half of the dnaC(D) activity recovered was was applied to a 0.5 X 20-cm column of BioGel A-5m agarose associated with dnaB. With half the amount of dnaC(D) gene equilibrated with 0.5 mM ATP, 1.0 mM MgCl2, 0.05 mg/ml of product and the same amount of dnaB gene product, all of bovine serum albumin, 30 mM potassium phosphate (pH 6.0), the dnaC(D) activity was associated with the dnaB activity and 10 mM dithiothreitol at room temperature. The column was (Fig. iF). developed with the same buffer at room temperature, and 0.1 ml Gel filtration of dnaB, dnaC(D), and ATP was affected by fractions were collected in tubes on ice containing 1 nmol of ATP. the addition of OX174 DNA. Under conditions as described Fractions were assayed as described in Materials and Methods for in Fig. 1A but with 6 nmol of oX174 DNA in the reaction, and complementing activities. (B) A dnaB (0) dnaC(D) (0) about 40% of the dnaB activity was excluded from the column was filtered reaction mixture minus dnaB gene product through and 60% eluted at its expected partially included position. an A-5m agarose column as described in part A. (C) A reaction of the was excluded, 30% mixture minus dnaC(D) gene product was filtered as described About 17% dnaC(D) activity in part A. (D) A reaction mixture minus ATP was filtered as eluted along with dnaB gene product, and 53% was fully described in part A but with ATP omitted from the column included.§ It was not clear whether dnaB was aggregated, buffer. (E) A reaction mixture containing dnaB (1.2 U), dnaC(D) associated with DNA directly, or associated with dnaC(D) (1.5 U), 0.5 mM ATP, 1 mM MgCl2, 30 mM potassium phosphate gene product and thus associated indirectly with DNA. (pH 6.0), 10 mM dithiothreitol, and 0.05 mg/ml of bovine serum albumin was incubated 30 min at 300. The reaction was then § Under conditions described in footnote t, at pH 7.5, neither filtered as described in part A but with ATP omitted from the dnaB nor dnaC(D) activity was in the excluded volume when the column buffer. (F) A reaction mixture as described in part A two proteins were mixed with 6 nmol of oX174 DNA and sub- containing dnaB (1.2 U) and dnaC(D) (0.75 U) was filtered as jected to gel filtration. Part of the dnaC(D) activity was asso- described in part A. ciated with the dnaB gene product and part was free. Downloaded by guest on October 1, 2021 Proc. Nat. Acad. SC?,'. USA 72 (1975) Interaction of dnaB and dnaC(D) Gene Products 923

A. dnaB gene product 0 24 zo I I :t is 1.5

12 1.0 3 ImcU Jo 6 05 DO a 10 20

Timee of Incubation (min) B. dnaC gene product FIG. 3. Rapid inhibition of dnaB ATPase by dnaC(D) gene 20 60 product. ATPase reaction mixtures were as described in Materials E and Methods. After the indicated times of incubation at 25°, _a0 15 45 & zl 'iPi production was measured catalyzed by dnaB gene product 0L> (0.05 U) alone (0); by dnaB (0.05 U) with dnaC(D) (0.2 U) _ O 2 10 30 added at 0 time (A); or by dnaB (0.05 U) with dnaC(D) (0.2 U) Oc, 0 added 8 min after the start of the incubation (0). . iC 5 15 X an extensive purification to apparent homogeneity (21). 0e 4 8 12 16 20 24 Fig. 2B shows that dnaC(D) complementing activity co- Fraction Number sedimented through a glycerol gradient with the inhibition of dnaB. ATPase. In addition, inhibition of ATPase activity FIG. 2. Association of ATPase with dnaB and inhibition of and dnaC(D) complementing activity were inactivated by ATPase activity with dnaC(D) gene product. (A) dnaB gene heat at the same rate (50% in 3 min at 55°). Also, treatment product was sedimented through a 5.2-mil, 15-35% glycerol of the dnaC(D) gene product preparation with N-ethyl- gradient containing 0.2 M KCJ, 20 mM potassium phosphate (pH 7.5), 1 mM EDTA, and 1 mM dithiothreitol for 9 hr at maleimide resulted in inactivation of both dnaC(D) com- 50,000 rpm in a Spinco SW 50.1 rotor. Twenty-five fractions were piementing activity [as previously shown (16) j and inhibition collected and assayed for dnaB complementing activity (0), of dnaB ATPase activity. ATPase activity in the absence of DNA (0), ATPase in the The inhibition by dnaC(D) of dnaB ATPase activity oc- presence of 2 nmol of OX174 DNA (0), ATPase activity by 10 curred rapidly. Fig. 3 shows that addition of dnaC(D) gene pA of each fraction in the absence of DNA in the presence of 0.2 U product to reactions in which dndB gene product was catalyz- of dnaC(D) gene product (A),, and ATPase activity by 10 ,A of ing the hydrolysis of ATP immediately prevented further each fraction in the, presence of 2 nmol of OX174 DNA and 0.2 U hydrolysis. This is consistent with the data shown above, of dnaC(D) (A). (B) dnaC(D) gene product was sedimented that the physical interaction of dnaB and dnaC(D) gene prod- through a 5.2-ml, 15-35% glycerol gradient as described above ucts occurred rapidly. for 27 hr at 60,000 rpm in a Spinco SW 65 rotor. 26 fractions Protection of Gene Product were collected and assayed for dnaC(D) complementing activity dnaC(D) from N-Ethylmaleimide (0) and inhibition of dnaB ATPase activity (0.1 U of dnaB) by Inactivation by dnaB Gene Product. As mentioned above, the 2 ul of each fraction (0). complementing activity of dnaC(D) gene product is in-

Inhibition of dnaB Ribonucleoside Triphosphatase Activity TABLE 1. Inhibition of dnaB ribonucleoside triphosphatase by dnaC(D) Gene Product. The dnaB gene product has asso- activity by dnaC(D) gene product ciated with it a ribonucleoside triphosphatase activity that is stimulated by single-stranded DNA but not by double- ATP GTP stranded DNA or RNA (21). The products of the reaction are Additions hydrolysis hydrolysis Pi and NDP. Table 1 shows that in the absence of DNA, (nmol of 32Pi/30 min) dnaB ATPase activity was inhibited by dnaC(D) gene product. - DNA GTPase activity was also inhibited, but 10 times higher con- dnaB 1.13 0.96 centrations of dnaC(D) were required; similarly, UTP dnaC(D) (0.20 U) <0. 02 <0.02 hydrolysis by dnaB was inhibited by high concentrations of dnaB + dnaC(D) (0. 03 U) 0.74 0.94 dnaC(D) gene product. In the presence of qX174 single- (0.1OU) 0.24 0.90 stranded DNA, dnaC(D) gene product only slightly inhibited (0.30 U) 0.04 0.48 dnaB ATPase or GTPase; similar results were obtained with + OX174 DNA fd DNA. dnaB 2.03 2.11 The experiment dnaC(D) (0.45 U) 0.07 0.07 following demonstrated that the inhibition dnaB + dnaC(D) (0. 15 U) 1.61 2.20 of the ATPase associated with dnaB was due to the action of (0.45 U) 1.45 1.40 dnaC(D) gene product on dnaB gene product. Fig. 2A shows that ATPase that cosedimented through a glycerol gradient Reaction mixtures were as described in Materials and Mdthods with dnaB activity was inhibited by dnaC(D) gene product. with 0.09 U of dnaB gene product, 0.5 nmol of 4X174 viral DNA, The dnaB complementing activity and ATPase activity h'-32P]ATP (130 cpm/pmol), or [y-32P]GTP (280 cpm/pmol), were previously shown to copurify over the last 20-fold of where indicated, and dnaC(D) gene product as indicated. Downloaded by guest on October 1, 2021 924 Biochemistry: Wickenr and Hurwitz Proc. Nat. Acad. Sci. USA 72 (1975) TABLE 2. Protection of dnaC(D) gene product from (under conditions as described in Materials and Methods with N-ethylmaleimide inactivation by dnaB gene product 1 mM dithiothreitol). The addition of dnaC(D) (0.07 U) in the absence of N-ethylmaleimide inhibited hydrolysis 83%; dnaC(D) the addition of dnaC(D) (0.07 U) treated with N-ethylmale- comple- imide prior to its addition to the ATPase assay mixture in- menting hibited hydrolysis 6%. The addition of N-ethylmaleimide (10 activity mM final concentration) after the start of ATPase reactions (pmol/20 % of Additions Treatment min at 300) control containing dnaB and dnaC(D) gene products had no effect on the inhibition of ATP hydrolysis catalyzed by dnaC(D). The dnaC(D) Dithiothreitol + dnaB ATPase activity after a 30 min incubation was inhibited N-ethylmaleimide 39.2 100 91 and 79% when N-ethylmaleimide was added at 1 and 10 dnaC(D) N-Ethylmaleimide 1.0 2.5 min, respectively, after the start of the reaction. dnaC(D) + ATP " 0.4 1.0 Thus, dnaB and dnaC(D) gene products mutually affect one dnaC(D) + dnaB " 2.5 6.4 to one another, dnaC(D) + dnaB another in reactions involving ATP; they bind + ATP 33.5 86 the ATPase of dnaB is inhibited by dnaC(D), and the N- dnaC(D) + dnaB ethylmaleimide sensitivity of dnaC(D) is prevented by dnaB + dATP, dTTP, gene product. dGTP, dCTP, DISCUSSION UTP, GTP, CTP, aB-methylene- The dnaB and dnaC(D) gene products and ATP interact ATP, or fly- physically and functionally in vitro. It is not known if this methylene-ATP " 0.5-1.0 1.5-2.5 interaction occurs in vivo. However, if the two proteins interact physiologically, then (a) dnaB gene product would be impli- Mixtures (0.02 ml) contained dnaC(D) (0.2 U), 30 mM potas- cated in initiation of replication since dnaC(D) sium phosphate (pH 6.5), 0.5 mM dithiothreitol, 1 mM MgC12, is required for this process (3), or (b) dnaC(D) gene product 0.05 mg/ml of bovine serum albumin, and 0.5 mM ATP or other would. be implicated in chromosome elongation since dnaB nucleotides and dnaB (0.3 U) where indicated. They were incu- is required for elongation (2), or (c) both proteins would be bated with 10 mM N-ethylmaleimide or the combination of 10 initiation and elongation. mM N-ethylmialeimide and 50 mM dithiothreitol as indicated. implicated in both chromosome After 20 min at 250, mixtures containing N-ethylmaleimide were Consistent with the possibility that these two proteins func- adjusted to 50 mM dithiothreitol. Samples (5 Al) were then tion jointly, some dnaC(D) mutants stop DNA synthesis assayed for dnaC(D) complementing activity as described in immediately at the elevated temperature (J. Wechsler, per- Materials and Methods. sonal communication). This observation suggests that dnaC(D), like dnaB, is involved in chromosome elongation. E. coli DNA replication, was In addition to being required for activated by N-ethylmaleimide. This inactivation pre- are required for con- product and ATP (Table 2). Neither both dnaB and dnaC(D) gene products vented by dnaB gene version in vitro of OX174 single-stranded DNA to duplex DNA of these components alone protected dnaC(D) complementing in combination with eight other purified proteins: dnaG gene activity nor could other nucleoside triphosphates be sub- from product, DNA polymerase III, DNA elongation factors I and stituted for ATP. The extent of protection of dnaC(D) and DNA factors X, Y, the ratio of II, DNA binding protein, replication inactivation by N-ethylmaleiimide depended on Z DNA by this reconstituted system of on the concentration and (23).11 synthesis dnaB to dnaC(D) gene product and also proteins requires ATP specifically (23). Three partial re- of ATP. At higher ratios of dnaB to dnaC(D) gene product, actions have been described that may contribute to this ATP higher concentrations of ATP were required to protect dna- of requirement. One is the interaction of dnaB and dnaC(D) C(D) from inactivation, possibly because of hydrolysis gene products demonstrated here. A second is the ribonucleo- ATP by the excess dnaB; at low ratios of dnaB to dnaC(D) of dnaB gene product that of side triphosphatase activity gene product there was only partial protection dnaC(D) not dNTPs A third is the elongation and the utilizes NTPs but (21). from' N-ethylmaleimide inactivation, protection of RNA- or DNA-primed single-stranded DNA that requires required lower concentrations of ATP. The protection of reactions of dnaB ATP or dATP.1' It is possible that other partial dnaC(D) against N-ethylmaleimide inactivation by the system will be found to involve the of DNA. The the 4X174 DNA-synthesizing gene product was influenced by presence ATP. addition of 4X174 or fd DNA increased the inactivation of reconstituted 4X174 DNA system it the of dnaB and With the replicating dnaC(D) by N-ethylmaleimide in presence is now possible to further dissect the system into its partial ATP; DNA alone or with ATP did not protect dnaC(D) from inactivation. factor Y The addition of N-ethylmaleimide to reactions used for the ¶1 The factor previously referred to as DNA replication dnaB and dna- has been resolved into two protein fractions; they will now be measurement of ATPase activity [containing Y and Z. DNA Materials and referred to as replication factors binding protein, C(D) gene products and ATP as described in elongation factors I and II, and replication factors X, Y, and Z Methods] did not reverse the inhibition of dnaB ATPase by are as yet undefined by genetic loci. dnaC(D). This is consistent with the observation that the 1I We have previously reported that the elongation reaction combination of dnaB and ATP protect dnaC(D) from N- catalyzed by DNA polymerase III and DNA elongation factors ethylmaleimide inactivation. In these experiments the rate of I and II requires ATP or dATP (24). Wickner and Kornberg (25) ATP hydrolysis by dnaB (0.06 U) was 1.6 nmol in 30 min at have reported a similar reaction in the presence of copolymerase 300 in the presence or absence of 10 mM N-ethylmaleimide III* and DNA polymerase III* that requires ATP. Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Interaction of dnaB and dnaC(D) Gene Products 925

reactions. So far the reaction has been resolved into two steps. 4. Wechsler, J. A. & Gross, J. D. (1971) Mol. Gen. Genet. 113, Step one requires dnaB and dnaC(D) gene products in addition 273-284. 5. Sakai, H., Hashimoto, S. & Komano, T. (1974) J. Bacteriol. to DNA binding protein and replication factors X, Y, and Z, 119, 811-820. ATP, and OX174 DNA, and involves reactions prior to dNMP 6. Filip, C., Allen, J. S., Gustafson, R. A., Allen, R. G. & incorporation; dnaG gene product, DNA polymerase III, and Walker, J. R. (1974) J. Bacteriol. 119, 443-449. DNA elongation factors I and II are not required during 7. Beyersmann, D., Messer, W. & Schlicht, M. (1974) J. Bacteriol. 118, 783-789. step 1 but are for dNMP the required incorporation during 8. Wada, C. & Yura, T. (1974) Genetics 77, 199-220. second step after the addition of dNTPs (23). Recently -we 9. Pauling, C. & Hamm, L. (1968) Proc. Nat. Acad. Sci. USA have physically separated these two steps (results to be 60, 1495-1502. published elsewhere). Incubation of the components required 10. DeLucia, P. & Cairns, J. (1969) Nature 224, 1164. for step 1 followed by BioGel A-5m gel filtration resulted in 11. Gefter, M. L., Hirota, Y., Kornberg, T., Wechsler, J. & Barnoux, C. (1971) Proc. Nat. Acad.- Sci. USA 68, 3150- the isolation of a complex associated with 4X174 DNA in the 3153. excluded volume. The addition of the components required 12. Nfisslein, V., Otto, B., Bonhoeffer, F. & Schaller, H. (1971) for step 2 to this complex resulted in dNMP incorporation. Nature New Biol. 234, 285- 286. Analysis of the complex isolated after gel filtration indicated 13. Konrad, E. B.-, Modrich, P. & Lehman, I. R. (1973) J. Mol. Biol. 77, 519-529. that 25-50% of the dnaB gene recovered was asso- product 14. Gottesman, M. M., Hicks, M. L. & Gellert, M. (1973) J. ciated with the OX174 DNA in the excluded volume; the rest Mol. Biol. 77, 531-547. was partially included in the gel. In contrast, <5% of the 15. Konrad, E. B. & Lehman, I. R. (1974) Proc. Nat. Acad. $ci. dnaC(D) gene product recovered was associated with the USA 71, 2048-2051. DNA; all detectable activity was associated with dnaB in 16. Wickner, S., Berkower, I., Wright, M. & Hurwitz, J. (1973) Proc. Nat. Acad. Sci. USA 70, 2369-2373. the partially included volume or free in the fully included 17. Wickner, S., Wright, M. & Hurwitz, J. (1973) Proc. Nat. volume. Thus, it is possible that the complex of dnaB and Acad. Sci. USA 70, 1613-1618. dnaC(D) gene products participates in a reaction resulting in 18. Wright, M., Wickner, S. & Hurwitz, J. (1973) Proc. Nat. the transfer of dnaB to 4,X174 DNA and the concomitant Acad. Sci. USA 70, 3120-3124. release of dnaC(D) gene product. 19. Wickner, S., Wright, M., Berkower, I. & Hurwitz, J. (1974) DNA Replication, ed. Wickner, R. B. (Marcel Dekker, Inc., New York). This research was carried out while S.W. was a guest scientist 20. Wechsler, J. A. (1973) DNA Synthesis In Vitro, eds. Wells, in Dr. W. Parks' laboratory. The authors are indebted to Dr. R. D. & Inman, R. B. (University Park Press, Baltimore, Parks for his hospitality. The work was supported in part by Md.). grants from the National Institutes of Health, National Science 21. Wickner, S., Wright, M. & Hurwitz, J. (1974) Proc. Nat. Foundation and the American Cancer Society. S.W. is a National Acad. Sci. USA 71, 783-787. Institutes of Health Postdoctoral Fellow. 22. Conway, T. W. & Lipmann, F. (1964) Proc. Nat. Acad. Sci. USA 52, 1462-1469. 23. Wickner, S. & Hurwitz, J. (1974) Proc. Nat. Acad. Sci. USA 1. Hirota, Y., Ryter, A. & Jacob, F. (1968) Cold Spring 71, 4120-4124. Harbor Symp. Quant. Biol. 33, 677-693. 24. Hurwitz, J. & Wickner, S. (1974) Proc. Nat. Acad. Sci. USA 2. Kohiyama, M. (1968) Cold Spring Harbor Symp. Quant. 71, 6-10. Biol. 33, 317-324. 25. Wickner, W. & Kornberg, A. (1973) Proc. Nat. Acad. Sci. 3. Carl, P. L. (1970) Mol. Gen. Genet. 109, 107-122. USA 70, 3679-3683. Downloaded by guest on October 1, 2021