Spectroscopic demonstration of a linkage between the kinetics of NTP hydrolysis and the conformational state of the recA-single-stranded DNA complex.
We recently constructed a mutant recA protein in which His-163 was replaced by a tryptophan residue; the [H163W]recA protein is functionally identical to the wild-type protein, and the Trp-163 side chain serves as a reporter group for the conformational transitions of the [H163W]recA-single-stranded DNA (ssDNA) complex. We have now examined the fluorescence properties of the [H163W]recA-ssDNA complex in the presence of a series of alternate nucleoside triphosphate cofactors. Under standard conditions (pH 7.5), ATP (S0.5 = 70 microM) and purine riboside triphosphate (PTP) (S0.5 = 110 microM) effect a 44% decrease in Trp-163 fluorescence and are active as cofactors for the DNA strand exchange reaction. In contrast, ITP (S0.5 = 400 microM) elicits only a 20% decrease in Trp-163 fluorescence (a level identical to that observed with the nucleoside diphosphates ADP, PDP, and IDP) and is inactive as a strand exchange cofactor. If the S0.5 (PTP) is increased to 130 microM (by increasing the pH of the reaction solution), the PTP-mediated quenching of Trp-163 fluorescence decreases to 20%, and PTP becomes inactive as a strand exchange cofactor. These results provide direct evidence for a linkage between the S0.5 value of a nucleoside triphosphate and the conformational state of the recA-ssDNA complex, with an S0.5 of 100-120 microM or lower required for stabilization of the strand exchange-active conformation.
Stole
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Department of Biochemistry, Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205, USA.
Bryant
F R
FR
D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017354:Point Mutation; D011685:Purine Nucleotides / Q000378:metabolism*; D011938:Rec A Recombinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan
D004277:DNA, Single-Stranded; D011685:Purine Nucleotides; D011994:Recombinant Proteins; D006639:Histidine; D014364:Tryptophan; D011938:Rec A Recombinases