Disassembly of Escherichia coli RecA E38K/DeltaC17 nucleoprotein filaments is required to complete DNA strand exchange.
Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/DeltaC17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.5. It will also form filaments on DNA and promote DNA pairing. However, below pH 7.3, ATP hydrolysis is completely uncoupled from extended DNA strand exchange. The products of extended DNA strand exchange do not form. At the lower pH values, disassembly of RecA E38K/DeltaC17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/DeltaC17 filaments improves at pH 8.5, whereas complete DNA strand exchange is also restored. Under these sets of conditions, a tight correlation between filament disassembly and completion of DNA strand exchange is observed. This correlation provides evidence that RecA filament disassembly plays a major role in, and may be required for, DNA strand exchange. A requirement for RecA filament disassembly in DNA strand exchange has a variety of ramifications for the current models linking ATP hydrolysis to DNA strand exchange.
Britt
Rachel L
RL
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Haruta
Nami
N
Lusetti
Shelley L
SL
Chitteni-Pattu
Sindhu
S
Inman
Ross B
RB
Cox
Michael M
MM
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008854:Microscopy, Electron / Q000379:methods; D009154:Mutation; D009698:Nucleoproteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000378:metabolism*; D013329:Structure-Activity Relationship
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D009698:Nucleoproteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases