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Interaction of Escherichia Coli Dnab and Dnac(D) Gene Products in Vitro

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Proc. Nat. Acad. Sci. USA Vol. 72, No. 3, pp. 921-925, March 1975 Interaction of Escherichia coli dnaB and dnaC(D) Gene 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 genes 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 polymerase 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.
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  • Ordered Association of Helicase Loader Proteins with the Bacillus Subtilis Origin of Replication in Vivo

    Ordered Association of Helicase Loader Proteins with the Bacillus Subtilis Origin of Replication in Vivo

    Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Smits, Wiep Klaas, Alexi I. Goranov, and Alan D. Grossman. “Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo.” Molecular Microbiology 75.2 (2010): 452–461. As Published http://dx.doi.org/10.1111/j.1365-2958.2009.06999.x Publisher Wiley Blackwell (Blackwell Publishing) Version Author's final manuscript Citable link http://hdl.handle.net/1721.1/73616 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike 3.0 Detailed Terms http://creativecommons.org/licenses/by-nc-sa/3.0/ NIH Public Access Author Manuscript Mol Microbiol. Author manuscript; available in PMC 2011 January 1. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Mol Microbiol. 2010 January ; 75(2): 452±461. doi:10.1111/j.1365-2958.2009.06999.x. Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo Wiep Klaas Smits, Alexi I. Goranov, and Alan D. Grossman* Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Summary The essential proteins DnaB, DnaD, and DnaI of Bacillus subtilis are required for initiation, but not elongation, of DNA replication, and for replication restart at stalled forks. The interactions and functions of these proteins have largely been determined in vitro based on their roles in replication restart. During replication initiation in vivo, it is not known if these proteins, and the replication initiator DnaA, associate with oriC independently of each other by virtue of their DNA binding activities, as a (sub)complex like other loader proteins, or in a particular dependent order.