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Activation of a recombinase-deficient mutant recA protein with alternate nucleoside triphosphate cofactors
1Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205.
Abstract:
We recently described two mutant recA proteins, (G160N)recA and (H163A)recA, which have full single-stranded DNA-dependent ATP hydrolysis activity but which are unable to promote the ATP-dependent strand exchange reaction under standard reaction conditions (pH 7.5). These mutant proteins, however, are able to promote strand exchange at pH 6.0 to 6.8. Here we show that this activation correlates with a pH-dependent decrease in the S0.5 value for ATP, with the (H163A)recA protein becoming active in strand exchange at pH values where the S0.5(ATP) decreases below 100 microM. We also show that the (H163A)recA protein is active in strand exchange over the range of pH 6.0-8.2 if dATP (or ddATP) is used in place of ATP as a cofactor; dATP is hydrolyzed by (H163A)recA protein at the same rate as ATP but has an S0.5 value lower than 100 microM across this pH range. These results are discussed with regard to the general significance of the S0.5 value in determining whether a nucleoside triphosphate will be able to stabilize the recA-single-stranded DNA filament in the strand exchange active conformational state.
Insights
Mutant recA proteins show pH-dependent strand exchange activity, activated when ATP
Area of Science:
- Molecular Biology
- Biochemistry
- Protein Engineering
Background:
- RecA protein is crucial for DNA repair and recombination.
- Mutant recA proteins (G160N)recA and (H163A)recA exhibit altered functionality.
- Standard conditions (pH 7.5) inhibit strand exchange for these mutants.
Purpose of the Study:
- Investigate the pH-dependent activation of mutant recA proteins in strand exchange.
- Determine the role of ATP's S0.5 value in recA filament stabilization.
- Explore alternative cofactor effects on mutant recA activity.
Main Methods:
- Enzyme kinetics assays measuring ATP hydrolysis and strand exchange.
- pH variation studies to assess protein activity.
- Cofactor substitution experiments using dATP and ddATP.
Main Results:
- Strand exchange activity for (G160N)recA and (H163A)recA is restored at pH 6.0-6.8.
- Activation correlates with a decrease in S0.5(ATP) below 100 microM for (H163A)recA.
- (H163A)recA regains strand exchange activity across pH 6.0-8.2 with dATP/ddATP, which have low S0.5 values.
Conclusions:
- The S0.5 value of nucleoside triphosphates is critical for stabilizing the active recA-DNA filament.
- pH influences recA activity by modulating the cofactor's S0.5 value.
- Understanding these parameters aids in designing or understanding recA-like functions.