Loop 2 in Saccharomyces cerevisiae Rad51 protein regulates filament formation and ATPase activity.
Previous studies showed that the K342E substitution in the Saccharomyces cerevisiae Rad51 protein increases the interaction with Rad54 protein in the two-hybrid system, leads to increased sensitivity to the alkylating agent MMS and hyper-recombination in an oligonucleotide-mediated gene targeting assay. K342 localizes in loop 2, a region of Rad51 whose function is not well understood. Here, we show that Rad51-K342E displays DNA-independent and DNA-dependent ATPase activities, owing to its ability to form filaments in the absence of a DNA lattice. These filaments exhibit a compressed pitch of 81 A, whereas filaments of wild-type Rad51 and Rad51-K342E on DNA form extended filaments with a 97 A pitch. Rad51-K342E shows near normal binding to ssDNA, but displays a defect in dsDNA binding, resulting in less stable protein-dsDNA complexes. The mutant protein is capable of catalyzing the DNA strand exchange reaction and is insensitive to inhibition by the early addition of dsDNA. Wild-type Rad51 protein is inhibited under such conditions, because of its ability to bind dsDNA. No significant changes in the interaction between Rad51-K342E and Rad54 could be identified. These findings suggest that loop 2 contributes to the primary DNA-binding site in Rad51, controlling filament formation and ATPase activity.
Zhang
Xiao-Ping
XP
Department of Microbiology, University of California, Davis, CA 95616-8665, USA.
Galkin
Vitold E
VE
Yu
Xiong
X
Egelman
Edward H
EH
Heyer
Wolf-Dietrich
WD
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D045643:DNA Repair Enzymes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D051135:Rad51 Recombinase / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure
D029701:Saccharomyces cerevisiae Proteins; D000244:Adenosine Diphosphate; D004247:DNA; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes