DNA helicase RepA: cooperative ATPase activity and binding of nucleotides.
The steady-state kinetic parameters of the ATPase activity of the homohexameric DNA helicase RepA and the binding of the fluorescent analogue epsilonADP to RepA have been studied. ssDNA stimulates RepA ATPase activity optimally at acidic pH 5.3-6.0. The sigmoidal kinetic curves in both the absence and presence of ssDNA show strong positive cooperativity for ATP hydrolysis, with oligonucleotides longer than 10mer optimal for ssDNA-stimulated ATPase activity. Fluorescence titrations show that, at 25 degrees C and in the absence of DNA, the binding of epsilonADP to RepA is biphasic with three high (K(1) = 1.54 x 10(6) M(-1)) and three low (K(2) = 4.71 x 10(4) M(-)(1)) affinity binding sites differing by 30-40-fold in binding constants. In the absence of cofactors, RepA melts cooperatively at T(m) = 65.8 +/- 0.1 degrees C and is more stable in the presence of ATPgammaS, T(m) = 68.1 +/- 0.2 degrees C (DeltaDeltaG 0.95 kcal/mol), than in the presence of ADP, T(m) = 66. 5 +/- 0.1 degrees C (DeltaDeltaG 0.29 kcal/mol), indicating that the additional phosphate group in ATPgammaS has a significant influence on RepA structure. A model is proposed in which individual subunits of RepA sequentially and cooperatively perform a multistep ATP hydrolytic cycle.
Xu
H
H
Institut für Kristallographie, Freie Universität Berlin, Takustrasse 6, D-14195 Berlin, Germany.
Frank
J
J
Niedenzu
T
T
Saenger
W
W
D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D015534:Trans-Activators
D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; C015195:1,N(6)-ethenoadenosine diphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases