Mutational analyses of the human Rad51-Tyr315 residue, a site for phosphorylation in leukaemia cells.
The human Rad51 protein, which plays a central role in homologous recombination, catalyses homologous pairing. The Rad51-Tyr315 residue is known to be constitutively phosphorylated in leukaemia cells and is thought to reside within the subunit-subunit interface of the Rad51 filament. To study the function of the Tyr315 residue, we purified five Rad51 mutants, Y315D, Y315E, Y315R, Y315A and Y315F, in which the Tyr315 residue was replaced by Asp, Glu, Arg, Ala and Phe, respectively. Biochemical studies of these Rad51 mutants revealed that the Y315D and Y315E mutants are defective in homologous pairing due to their impaired ssDNA binding, but their dsDNA binding remained unaffected. The Y315D, Y315E and Y315R mutants are defective in dsDNA unwinding, which depends on Rad51-filament formation, suggesting that these mutants are defective in filament formation on dsDNA. Therefore, the Rad51-Tyr315 residue plays important roles in ssDNA binding and filament formation.
Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Yoshimasa
Y
Takizawa
Takashi
T
Kinebuchi
Wataru
W
Kagawa
Shigeyuki
S
Yokoyama
Takehiko
T
Shibata
Hitoshi
H
Kurumizaka
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007938:Leukemia / Q000378:metabolism; D010766:Phosphorylation; D051135:Rad51 Recombinase; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014443:Tyrosine; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases; D004265:DNA Helicases