Inhibition of RecA Protein Function by the RdgC Protein from Escherichia coli

Julia C Drees,Sindhu Chitteni-Pattu,Darrell R Mccaslin,Ross B Inman,Michael M Cox

doi:10.1074/jbc.m513592200

Julia C Drees, Sindhu Chitteni-Pattu + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m513592200

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Abstract

The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI protein. RdgC protein can inhibit DNA strand exchange catalyzed by RecA nucleoprotein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoprotein filaments. RdgC protein binds to single-stranded and double-stranded DNA, and the protein can be visualized on DNA using electron microscopy. RdgC protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.

Highlights

The activities of RecA protein must be regulated in the cell to target RecA to locations where it is needed and to avoid aberrant DNA transactions
The RdgC protein is important in strains lacking PriA, the replication restart protein, because it alleviates a toxic effect of the RecFOR proteins [26]
RdgC Protein Inhibits RecA Protein Activities—We first surveyed the effects of RdgC protein on a series of classic RecA functions

Summary

EXPERIMENTAL PROCEDURES

Enzymes—The E. coli wild-type RecA protein and the RecA⌬C17 mutant were purified as described previously [13]. The E. coli SSB protein was purified as described before [29]. Media, and Reagents—R buffer contained 20 mM Tris-HCl (80% cation, pH 7.5), 1 mM DTT, 0.1 mM EDTA, and 10% (w/v) glycerol. TAE buffer contained 40 mM Tris-OAc (80% cation) and 1 mM EDTA. Laemmli sample buffer contained 250 mM Tris-Cl, pH 6.8, 4% SDS, 20% (w/v) glycerol, 10% ␤-MeOH, and 0.1% (w/v) bromphenol blue. TBE buffer contained 90 mM Tris borate and 10 mM EDTA. Fluorescence polarization buffer contained 25 mM Tris-OAc (80% cation), 5% (w/v) glycerol, 3 mM potassium glutamate, 10 mM Mg(OAc)2, 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin, and 1 mM DTT

RdgC Inhibition of RecA

RESULTS

DISCUSSION