Magnesium ion-dependent activation of the RecA protein involves the C terminus.
Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mm Mg(2+) in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg(2+)) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg(2+) results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg(2+) and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg(2+) levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.
Lusetti
Shelley L
SL
Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA.
Shaw
Jeffrey J
JJ
Cox
Michael M
MM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism
D029968:Escherichia coli Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases; D008274:Magnesium