Effect of ions and nucleotides on the interactions of yeast Rad51 protein with single-stranded oligonucleotides.
Rad51 protein is a eukaryotic homologue of RecA protein that is essential for homologous recombination. We developed a simple procedure for purifying yeast Rad51 protein, characterized its interaction with DNA, and compared it with those of RecA from Escherichia coli and Rad51 from higher eukaryotes. Fractionation of crude extract with 0.2% polyethylenimine eliminated contaminant proteins and nucleic acids, which can perturb the subsequent purification steps. Binding of Rad51 to single-stranded DNA was detected in solution by measuring the fluorescence anisotropy of a fluorescein probe attached to the 5' end of the oligonucleotides. The interaction was stabilized by ATP, as is that of RecA, but was neither stabilized by a non-hydrolysable analog of ATP, nor destabilized by ADP, unlike the interaction of RecA. This character was very similar to that of Xenopus XRad51.1, although the binding of yeast Rad51 to DNA was more sensitive to Mg(2+) ion in both the presence and absence of ATP, and was optimal at 5--10 mM Mg(2+). The dissociation of Rad51 protein from DNA is not, therefore, favored by the hydrolysis of ATP to ADP, in contrast to that of RecA. On the other hand, the high DNA-binding state of the Rad51-DNA complex promoted by ATP appeared to be short-lived. These features may be linked to the lower activity of Rad51 and the fact that Rad51 activity does not require the hydrolysis of ATP.
Department of Chemistry, College of Natural Sciences, Yeungnam University, Kyoungsan City, Kyoung-buk, 712-749 Republic of Korea.
J M
JM
Kim
F
F
Maraboeuf
S K
SK
Kim
A
A
Shinohara
M
M
Takahashi
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000838:Anions / Q000378:metabolism; D015153:Blotting, Western; D002413:Cations, Divalent / Q000378:metabolism; D011232:Chemical Precipitation; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D011094:Polyethyleneimine / Q000378:metabolism; D011485:Protein Binding; D051135:Rad51 Recombinase; D011938:Rec A Recombinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015003:Yeasts
D000838:Anions; D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011094:Polyethyleneimine; D051135:Rad51 Recombinase; D011938:Rec A Recombinases; D008274:Magnesium