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Purification Andproperties of the Y-Protein Specified By Proc. Nat. Acad. Sci. USA Vol. 70, No. 8, pp. 2215-2219, August 1973 Purification and Properties of the y-Protein Specified by Bacteriophage X: An Inhibitor of the Host RecBC Recombination Enzyme (bacteriophage X gami gene/RecBC DNase/exonuclease V) YOSHIYUKI SAKAKI, ALEXANDER E. KARU, STUART LINN, AND HARRISON ECHOLS Department of Molecular Biology and Department of Biochemistry, University of California, Berkeley, Calif. 94720 Communicated by Charles Yanofsky, May 9, 1973 ABSTRACT Previous experiments have indicated that double mutant JC6720; the Su recB21 mutant JC4695 (3, 7). the gam gene of bacteriophage X is responsible for an inhi- The X phage mutations used were: redX314, a mutation in bition of the RecBC DNase-an enzyme that is essential for the major host pathway of genetic recombination. We re- the structural gene for X-exonuclease (8); gam210 and gam37, port here experiments that define the inhibitor as the pro- nonsense and temperature-sensitive mutations, respectively, tein product of the gam gene ("1y-protein") and that char- in the gam gene (1); biol, a deletion-addition mutation that acterize the inhibition reaction with highly purified prep- has deleted most of the X recombination region including arations of e-protein and RecBC DNase. Genetic charac- terization was performed with partially purified fractions the gam gene (1); bio72, a deletion-addition mutation that prepared from cells infected with various X mutants. An ac- has deleted most of the X recombination region, but not the tivity that inhibits RecBC DNase was absent in extracts gam gene (1). All phage except bio72gam37 carried the cd857 prepared after infection by phage that carry nonsense or mutation, which renders the X cI protein thermolabile and deletion mutations in the gam gene; this activity was thus allows thermal induction of X prophage highly thermolabile in an extract prepared after infection (9). by phage that carry a temperature-sensitive mutation in DNA and Enzymes. E. coli ['H]DNA (5500-7500 cpm/ the gam gene. For biochemical characterization, the -y-pro- nmol) was prepared by the methods of Oishi (10), or Lehman tein has been purified more than 800-fold. This highly pur- ified preparation inhibited all of the known catalytic ac- (11). Phage T7 ['HIDNA (17,000 cpm/nmol) was prepared tivities associated with the RecBC enzyme, but exhibited according to Richardson (12); E. coli ['H]DNA with inter- no detectable DNase or ATPase activities by itself. These strand crosslinks introduced by treatment with 4,5',8-tri- findings are discussed in terms of their implications for methylpsoralen was prepared as described (13). regulation of genetic recombination and bacteriophage X development. The RecBC DNase used to monitor the purification of 7-protein ("Brij lysate preparation") was made as follows. The product of the gam gene of phage X participates in an A culture of E. coli JC6724 (6 to 8 X 108 cells per ml) was interesting and complex variety of host-virus interactions, harvested, lysed with Brijj and fractionated with strepto- including: (a) the capacity of X to grow (make a plaque) on a mycin sulfate and ammonium sulfate as described below for recA- recB+ host [Fec phenotype (1)]; (b) the failure of X the purification of y-protein. The ammonium sulfate fraction to grow on a host lysogenic for phage P2 [Spi phenotype (1, (1-ml) was layered on a gradient (11-ml) of 10-30% glycerol 2)]; and (c) the inhibition of the host RecBC recombination [in 10 mM Tris * HOl (pH 8.0), 10 mM MgC12, 5 mM 2-mer- pathway (3). A unifying hypothesis to explain these phenom- captoethanol, and 0.1 M KCl] and centrifuged for 16 hr at ena involves the postulate that phage X converts recB+ 40,000 rpm in a Spinco SW41 rotor. Fractions about 65% of recC+ cells to a phenotype characteristic of recB- or recC- the way through the gradient contained the RecBC DNase. mutants and that the gam gene of X is required for this pheno- All studies with purified 7-protein (Fraction VII) used highly typic conversion (see ref. 4 and Discussion). purified RecBC DNase [Fraction IX as described by Gold- Recent experiments have supported this postulate at a bio- mark and Linn (14)] unless otherwise indicated. chemical level: crude extracts from cells infected by gam+ Exonuclease I was the hydroxylapatite fraction, prepared phage exhibit strongly diminished activity of the protein and assayed according to Lehman and Nussbaum (15). Exo- product of the recB and recC genes-the RecBC DNase- nuclease II (homogeneous DNA polymerase) and the Sepha- whereas extracts from cells infected by gam- phage show dex G-100 fraction of Exonuclease III, prepared as described normal levels of RecBC DNase activity (5). Since this in- by Jovin et al. (16), were generously provided by Dr. D. hibitory effect could be demonstrated by mixing gam+ and Brutlag. E. coli B restriction endonuclease was the sucrose gam- extracts, the most likely explanation for the diminished gradient fraction (17). Pancreatic RNase and RNase U1, activity of the Recl3C DNase is an inhibition of enzyme activ- free of DNase, were provided by Dr. A. Blank. ity, mediated directly or indirectly by the product of the Assay for 7y-Proten measured its capacity to inhibit the gam gene ("vy-protein") (5, 6). activity of the RecBC enzyme. The preparation of 7-protein This report presents evidence that the inhibitor is the y- was "preincubated" with RecBC DNase, and then the sub- protein itself and describes the purification of the y-protein strate (usually double-stranded DNA) was added to measure and its effects on purified RecBC DNase. remaining activity of the RecBC enzyme. For assay for exo- MATERIALS AND METHODS nuclease activity with double-stranded DNA and "Brij lysate preparation" of RecBC DNase, the following procedure was Bacteriophage and Bacteria. The Escherichia coli strains used. Preincubation mixtures contained: 10 mM Tris HCl used and their relevant genetic characteristics were: the Rec+ (pH 8.0), 10 mM MgCl2, 10 mM 2-mercaptoethanol, 0.5 mM Endonuclease I- strain JC6724 and its related recB21 recC22 ATP, and 0.2-0.6 units of "Brij lysate preparation" of RecBC 2215 Downloaded by guest on September 29, 2021 2216 Biochemistry: Sakaki et at, Proc. Nat. Acad. Sci. USA 70 (1973) - - 0.30 A 1.0 la gem+ goam + 00 0.8 gam\\ .' \0 E w 0.6 I___ ' >1 -6 a *i0 0.4 2 z Id- 0o ~ ~~0\~~~°\ ~ 0 c c _1 _ I\\I\ _ LP 0 2 4 6 8 10 Minutes at 41°C 4 8 12 4 8 1.2 FIG. 2. Thermolability of Inhibitor II in an extract from a Fraction Number temperature-sensitive gam mutant. Cultures of JC4695 were FIG. 1. Sedimentation properties of inhibitors of the RecBC grown at 28° and infected with Xbio72 (at a multiplicity of 4) or DNase. The experiments of A and B used thermal induction of Xbio72gamts37 (at a multiplicity of 15). Cells were harvested 20 the gam+ and gam- prophages in the lysogens JC4695(XcI857 min after infection. Extracts were prepared and sedimented in redX314) (A) and JC4695(XcI857redX314gam210) (B). Cultures glycerol gradients as described in Fig. 1. Samples of the inhibitory were grown at 300 and thermally induced by raising the tempera- activity that sedimented more slowly than hemoglobin (Inhibitor ture to 420 for 15 min. Extracts were prepared and purified II) were incubated at 410 for the times indicated, and then as- through Fraction III, as described in Methods. The experiments sayed (at 290) for inhibition of RecBC DNase. (0- - -0), In- of C and D used infection of JC4695 by the gam+ and gam- dele- hibitor II activity from Xbio72gamts37; (@-*), Inhibitor II tion mutants Xbio72 (C) and Xbiol (D). Cultures were grown at 370 activity from Xbio72gam +. and infected at a multiplicity of 4 phage per bacterium. After 15 min, extracts were prepared as for A and B. Sedimentation was done in a Spinco SW50.1 rotor at 40 in a 20-40% glycerol gradient (14), 20% glycerol, about 1 unit (0.02-,Ag) of RecBC DNase, [in 20 mM Tris * HCl (pH 8.0)-0.1 M KCl-1 mM EDTA-10 mM ATP and y-protein as noted. After 10 min at 37°, 4 nmol of 2-mercaptoethanol]. For A and B, sedimentation was at 45,000 [in 0.06 ml of 10 mM Tris * HCl (pH 8.2)-10 mM rpm for 28 hr; for C and D, sedimentation was at 49,000 rpm for [8H]DNA was incubation was continued for 30 min at 18 hr. Fractions were collected, and aliquots (S-l) were assayed MgCl2] added; for inhibition of the RecBC DNase. Sedimentation was from 370; 0.05 ml of bovine-serum albumin (10 mg/ml) was then right to left. Arrows denote the position of an internal hemoglobin added, and the reaction was terminated by addition of 0.25 marker (s2o,, of 4.3, molecular weight of 64,000). (- - -), Uninhib- ml of 7% trichloroacetic acid. For ATPase reactions, [y-_2p]- ited RecBC DNase activity added to the assay; (0 0), RecBC ATP (500-1500 cpm/nmol) was used, and the 32p rendered DNase activity in the presence of the gradient fractions. nonadsorbable to Norit was determined as described (14). The single-strand endonuclease assay was performed as de- scribed above, using circular fd [3H]DNA and 0.7 units of DNase in a total volume of 0.12 ml. The y-protein was added exonuclease I (14). at levels noted, and the mixture was incubated at 370 for 10 Purification of y-Protein. For the preparation used for most min.
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