Complementation of one RecA protein point mutation by another. Evidence for trans catalysis of ATP hydrolysis.
The RecA residues Lys248 and Glu96 are closely opposed across the RecA subunit-subunit interface in some recent models of the RecA nucleoprotein filament. The K248R and E96D single mutant proteins of the Escherichia coli RecA protein each bind to DNA and form nucleoprotein filaments but do not hydrolyze ATP or dATP. A mixture of K248R and E96D single mutant proteins restores dATP hydrolysis to 25% of the wild type rate, with maximum restoration seen when the proteins are present in a 1:1 ratio. The K248R/E96D double mutant RecA protein also hydrolyzes ATP and dATP at rates up to 10-fold higher than either single mutant, although at a reduced rate compared with the wild type protein. Thus, the K248R mutation partially complements the inactive E96D mutation and vice versa. The complementation is not sufficient to allow DNA strand exchange. The K248R and E96D mutations originate from opposite sides of the subunit-subunit interface. The functional complementation suggests that Lys248 plays a significant role in ATP hydrolysis in trans across the subunit-subunit interface in the RecA nucleoprotein filament. This could be part of a mechanism for the long range coordination of hydrolytic cycles between subunits within the RecA filament.
Cox
Julia M
JM
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706-1544, USA.
Abbott
Stephen N
SN
Chitteni-Pattu
Sindhu
S
Inman
Ross B
RB
Cox
Michael M
MM
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D002384:Catalysis; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry; D008854:Microscopy, Electron; D008968:Molecular Conformation; D009154:Mutation; D017354:Point Mutation; D011938:Rec A Recombinases / Q000235:genetics* / Q000378:metabolism
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases; D008239:Lysine