Design and Evaluation of a Tryptophanless RecA Protein with Wild Type Activity

Inhibition of the Escherichia coli RecA protein: zinc(II), copper(II) and mercury(II) trap RecA as inactive aggregates

“…The poly(dT)-dependent ATPase activity of RecA can be characterized using a well-established singlestrand DNA-dependent enzyme coupled ATPase assay [38,39]. However, in this case, several key alterations had to be made.…”

“…First, all metal chelating agents, including EDTA and DTT (racemic mixture), were removed from the reaction solution. To accomplish this, the RecA protein was purified from its storage buffer [38] using size-exclusion chromatography. The removal of DTT and EDTA from the control reaction (no inhibitor) had no adverse effects on the reaction velocity or the ATP turnover number of the RecA (data not shown).…”

“…We conducted ATPase kinetic measurements essentially as described previously [38] at 37°C in buffer containing 25 mM Tris AE HOAc, pH 7.5, and 10 mM Mg(OAc) 2 . We found that the Zn AE L L-DTT complex clearly inhibits the RecA ATPase activity at P25 lM complex (Figs.…”

“…1(a) and 2). We separated the Initial hydrolysis rates were monitored at 37°C in aqueous buffer (pH 7.1) using a coupled-enzyme spectrophotometric assay [38,39]. The reaction solutions contained 1.0 lM RecA protein, 3 lMnts poly(dT), 3 mM ATP, 25 mM Tris AE HOAc, 10 mM Mg(OAc) 2 , 2.3 mM PEP, 5 U/mL pyruvate kinase, 5 U/mL lactic dehydrogenase, 2 mM NADH, 5% w/v glycerol, and the indicated concentrations of Zn AE L L-DTT or ZnCl 2 .…”

Bismuth–dithiol inhibition of the Escherichia coli rho transcription termination factor

“…Electron micrograph studies of rho and RecA show that the cores of these proteins are structurally similar [52]. We observed an apparent I 50 of 21 lM for BiBAL (3:1) in the RecA poly(dT)-dependent ATPase assay [53,54] suggesting that this mixture may also target other RecA superfamily proteins (e.g., DnaB, RuvB, T7 helicase). At this time we do not know whether this inhibition resulted from the aggregation of the RecA filament into inactive bundles or from a site specific interaction process.…”