Biochemical characterization of the human RAD51 protein. III. Modulation of DNA binding by adenosine nucleotides.
Adenosine nucleotides affect the ability of RecA small middle dotsingle-stranded DNA (ssDNA) nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination. Here we have determined that ADP and ATP/ATPgammaS affect the DNA binding and aggregation properties of the human RecA homolog human RAD51 protein (hRAD51). These studies have revealed significant differences between hRAD51 and RecA. In the presence of ATPgammaS, RecA forms a stable complex with ssDNA, while the hRAD51 ssDNA complex is destabilized. Conversely, in the presence of ADP and ATP, the RecA ssDNA complex is unstable, while the hRAD51 ssDNA complex is stabilized. We identified two hRAD51 small middle dotssDNA binding forms by gel shift analysis, which were distinct from a well defined RecA small middle dotssDNA binding form. The available evidence suggests that a low molecular weight hRAD51 small middle dotssDNA binding form (hRAD51 small middle dotssDNA(low)) correlates with active ADP and ATP processing. A high molecular weight hRAD51 small middle dotssDNA aggregate (hRAD51 small middle dotssDNA(high)) appears to correlate with a form that fails to process ADP and ATP. Our data are consistent with the notion that hRAD51 is unable to appropriately coordinate ssDNA binding with adenosine nucleotide processing. These observations suggest that other factors may assist hRAD51 in order to mirror RecA recombinational function.
Tombline
Gregory
G
Genetics and Molecular Biology Program, Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Heinen
Christopher D
CD
Shim
Kang-Sup
KS
Fishel
Richard
R
D000227:Adenine Nucleotides / Q000378:metabolism*; D015374:Biosensing Techniques; D002850:Chromatography, Gel; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D051135:Rad51 Recombinase
D000227:Adenine Nucleotides; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase