Multiple interactions with the Rad51 recombinase govern the homologous recombination function of Rad54.
In eukaryotes, Rad51 and Rad54 functionally cooperate to mediate homologous recombination and the repair of damaged chromosomes by recombination. Rad51, the eukaryotic counterpart of the bacterial RecA recombinase, forms filaments on single-stranded DNA that are capable of pairing the bound DNA with a homologous double-stranded donor to yield joint molecules. Rad54 enhances the homologous DNA pairing reaction, and this stimulatory effect involves a physical interaction with Rad51. Correspondingly, the ability of Rad54 to hydrolyze ATP and introduce superhelical tension into covalently closed circular plasmid DNA is stimulated by Rad51. By controlled proteolysis, we show that the amino-terminal region of yeast Rad54 is rather unstructured. Truncation mutations that delete the N-terminal 113 or 129 amino acid residues of Rad54 attenuate or ablate physical and functional interactions with Rad51 under physiological ionic strength, respectively. Surprisingly, under less stringent conditions, the Rad54 Delta129 protein can interact with Rad51 in affinity pull-down and functional assays. These results highlight the functional importance of the N-terminal Rad51 interaction domain of Rad54 and reveal that Rad54 contacts Rad51 through separable epitopes.
Raschle
Markus
M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115-5730, USA.
Van Komen
Stephen
S
Chi
Peter
P
Ellenberger
Tom
T
Sung
Patrick
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D004265:DNA Helicases; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D051135:Rad51 Recombinase; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D004274:DNA, Recombinant; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes