Purification and Properties of the γ-Protein Specified by Bacteriophage λ: An Inhibitor of the Host RecBC Recombination Enzyme

Abstract

Previous experiments have indicated that the gam gene of bacteriophage X is responsible for an inhibition of the RecBC DNase-an enzyme that is essential for the major host pathway of genetic recombination. We report here experiments that define the inhibitor as the protein product of the gam gene ("1y-protein") This report presents evidence that the inhibitor is the yprotein itself and describes the purification of the y-protein and its effects on purified RecBC DNase. MATERIALS AND METHODSBacteriophage and Bacteria. The Escherichia coli strains used and their relevant genetic characteristics were: the Rec+ Endonuclease I-strain JC6724 and its related recB21 recC22 2215 double mutant JC6720; the Su recB21 mutant JC4695 (3, 7). The X phage mutations used were: redX314, a mutation in the structural gene for X-exonuclease (8); gam210 and gam37, nonsense and temperature-sensitive mutations, respectively, in the gam gene (1); biol, a deletion-addition mutation that has deleted most of the X recombination region including the gam gene (1); bio72, a deletion-addition mutation that has deleted most of the X recombination region, but not the gam gene (1). All phage except bio72gam37 carried the cd857 mutation, which renders the X cI protein thermolabile and thus allows thermal induction of X prophage (9). DNA and Enzymes. E. coli ['H]DNA (5500-7500 cpm/ nmol) was prepared by the methods of Oishi (10) The RecBC DNase used to monitor the purification of 7-protein ("Brij lysate preparation") was made as follows.A culture of E. coli JC6724 (6 to 8 X 108 cells per ml) was harvested, lysed with Brijj and fractionated with streptomycin sulfate and ammonium sulfate as described below for the purification of y-protein. The ammonium sulfate fraction(1-ml) was layered on a gradient (11-ml) of 10-30% glycerol [in 10 mM Tris * HOl (pH 8.0), 10 mM MgC12, 5 mM 2-mercaptoethanol, and 0. Assay for 7y-Proten measured its capacity to inhibit the activity of the RecBC enzyme. The preparation of 7-protein was "preincubated" with RecBC DNase, and then the substrate (usually double-stranded DNA) was added to measure remaining activity of the RecBC enzyme. For assay for exonuclease activity with double-stranded DNA and "Brij lysate preparation" of RecBC DNase, the following procedure was used. Preincubation mixtures contained: 10 mM Tris HCl (pH 8.0), 10 mM MgCl2, 10 mM 2-mercaptoethanol, 0.5 mM ATP, and 0.2-0.6 units of "Brij lysate preparation" of RecBC

Interaction of Bacterial and Lambda Phage Recombination Systems in the X-Ray Sensitivity of Escherichia coli K-12

“…For example, a limited nuclease attack might be required for successful DNA repair while extensive DNA degradation could prevent the successful completion of repair. This possibility is consistent with recent results of Sakaki et al (27), who have purified the '-protein and shown that it inhibits all known activities of the RecBC nuclease.…”

Completion of DNA replication in Escherichia coli

“…Late-phase replication in phage lambda requires expression of the phage Gam protein, which targets and inactivates RecD and SbcDC in the host (41,42). Similar to the amplification of phage and plasmid DNA (36), we observed that gam expression results in an over-replication of the terminus region (Fig.…”

How RecBCD Enzyme and Chi Promote DNA Break Repair and Recombination: a Molecular Biologist's View

“…exo was shown to encode an exonuclease (79) slightly before the discovery of the RecBCD nuclease. From related genetic studies came the additional finding that the gam gene encodes a protein that inhibits the RecBCD nuclease (82) and allows the phage to circumvent the destructive power of the bacterium. Thus, wild-type inhibits the bacterial recombination pathway and relies on its own recombination pathway.…”