Kinetic analysis of pairing and strand exchange catalyzed by RecA. Detection by fluorescence energy transfer.
RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. In this study, we measured RecA-catalyzed pairing and strand exchange in solution by energy transfer between fluorescent dyes on the ends of deoxyribo-oligonucleotides. By varying the position of the dyes in separate assays, we were able to detect the pairing of single-stranded RecA filament with duplex DNA as an increase in energy transfer, and strand displacement as a decrease in energy transfer. With these assays, the kinetics of pairing and strand displacement were studied by stopped-flow spectrofluorometry. The data revealed a rapid, second order, reversible pairing step that was followed by a slower, reversible, first order strand exchange step. These data indicate that an initial unstable intermediate exists which can readily return to reactants, and that a further, rate-limiting step (or steps) is required to effect or complete strand exchange.
Bazemore
L R
LR
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510, USA.
Takahashi
M
M
Radding
C M
CM
D001483:Base Sequence; D002384:Catalysis; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism*; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D012235:Rhodamines; D011938:Rec A Recombinases