Yeast recombination factor Rdh54 functionally interacts with the Rad51 recombinase and catalyzes Rad51 removal from DNA.
The Saccharomyces cerevisiae RDH54-encoded product, a member of the Swi2/Snf2 protein family, is needed for mitotic and meiotic interhomologue recombination and DNA repair. Previous biochemical studies employing Rdh54 purified from yeast cells have shown DNA-dependent ATP hydrolysis and DNA supercoiling by this protein, indicative of a DNA translocase function. Importantly, Rdh54 physically interacts with the Rad51 recombinase and promotes D-loop formation by the latter. Unfortunately, the low yield of Rdh54 from the yeast expression system has greatly hampered the progress on defining the functional interactions of this Swi2/Snf2-like factor with Rad51. Here we describe an E. coli expression system and purification scheme that together provide milligram quantities of nearly homogeneous Rdh54. Using this material, we demonstrate that Rdh54-mediated DNA supercoiling leads to transient DNA strand opening. Furthermore, at the expense of ATP hydrolysis, Rdh54 removes Rad51 from DNA. We furnish evidence that the Rad51 binding domain resides within the N terminus of Rdh54. Accordingly, N-terminal truncation mutants of Rdh54 that fail to bind Rad51 are also impaired for functional interactions with the latter. Interestingly, the rdh54 K352R mutation that ablates ATPase activity engenders a DNA repair defect even more severe than that seen in the rdh54Delta mutant. These results provide molecular information concerning the role of Rdh54 in homologous recombination and DNA repair, and they also demonstrate the functional significance of Rdh54.Rad51 complex formation. The Rdh54 expression and purification procedures described here should facilitate the functional dissection of this DNA recombination/repair factor.
Chi
Peter
P
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Kwon
Youngho
Y
Seong
Changhyun
C
Epshtein
Anastasiya
A
Lam
Isabel
I
Sung
Patrick
P
Klein
Hannah L
HL
D000255:Adenosine Triphosphate / Q000378:metabolism; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D045643:DNA Repair Enzymes; D026942:DNA Topoisomerases; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D008741:Methyl Methanesulfonate / Q000378:metabolism; D046912:Multiprotein Complexes; D009153:Mutagens / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004271:DNA, Fungal; D004278:DNA, Superhelical; D046912:Multiprotein Complexes; D009153:Mutagens; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D008741:Methyl Methanesulfonate; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D026942:DNA Topoisomerases; C541336:RDH54 protein, S cerevisiae; D045643:DNA Repair Enzymes