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Originally published In Press as doi:10.1074/jbc.M109917200 on February 11, 2002

J. Biol. Chem., Vol. 277, Issue 17, 14434-14442, April 26, 2002
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Biochemical Characterization of the Human RAD51 Protein
III. MODULATION OF DNA BINDING BY ADENOSINE NUCLEOTIDES*

Gregory Tombline, Christopher D. Heinen, Kang-Sup Shim, and Richard FishelDagger

From the Genetics and Molecular Biology Program, Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Adenosine nucleotides affect the ability of RecA·single-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/ATPgamma S 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 ATPgamma S, 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·ssDNA binding forms by gel shift analysis, which were distinct from a well defined RecA·ssDNA binding form. The available evidence suggests that a low molecular weight hRAD51·ssDNA binding form (hRAD51·ssDNAlow) correlates with active ADP and ATP processing. A high molecular weight hRAD51·ssDNA aggregate (hRAD51·ssDNAhigh) 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.

* This work was supported by National Research Service Award Grant 5-T32-CA09678 (to G. T.) and National Institutes of Health Grant CA56542 (to R. F.).

Dagger To whom correspondence and reprint requests should be addressed: Kimmel Cancer Center, BLSB933, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 213-503-1346; Fax: 215-923-1098; E-mail: rfishel@ hendrix.jci.tju.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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