RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis.
The Escherichia coli SOS response to DNA damage is modulated by the RecA protein, a recombinase that forms an extended filament on single-stranded DNA and hydrolyzes ATP. The RecA K72R (recA2201) mutation eliminates the ATPase activity of RecA protein. The mutation also limits the capacity of RecA to form long filaments in the presence of ATP. Strains with this mutation do not undergo SOS induction in vivo. We have combined the K72R variant of RecA with another mutation, RecA E38K (recA730). In vitro, the double mutant RecA E38K/K72R (recA730,2201) mimics the K72R mutant protein in that it has no ATPase activity. The double mutant protein will form long extended filaments on ssDNA and facilitate LexA cleavage almost as well as wild-type, and do so in the presence of ATP. Unlike recA K72R, the recA E38K/K72R double mutant promotes SOS induction in vivo after UV treatment. Thus, SOS induction does not require ATP hydrolysis by the RecA protein, but does require formation of extended RecA filaments. The RecA E38K/K72R protein represents an improved reagent for studies of the function of ATP hydrolysis by RecA in vivo and in vitro.
Gruenig
Marielle C
MC
Department of Biochemistry, 433 Babcock Drive, University of Wisconsin, Madison, WI 53706, USA.
Renzette
Nicholas
N
Long
Edward
E
Chitteni-Pattu
Sindhu
S
Inman
Ross B
RB
Cox
Michael M
MM
Sandler
Steven J
SJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D011938:Rec A Recombinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D013014:SOS Response, Genetics / Q000528:radiation effects; D012697:Serine Endopeptidases / Q000378:metabolism; D014466:Ultraviolet Rays; D000099160:LexA Repressor Protein
D000255:Adenosine Triphosphate; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D011938:Rec A Recombinases; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein