| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 17, 14426-14433, April 26, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Genetics and Molecular Biology Program, Department of
Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson
University, Philadelphia, Pennsylvania 19107
RecA mediated homologous recombination requires
cooperative ATP binding and hydrolysis to assume and maintain an
active, extended DNA-protein (nucleoprotein) filament. Human RAD51
protein (hRAD51) lacks the magnitude of ATP-induced cooperativity and
catalytic efficiency displayed by RecA. Here, we examined hRAD51
binding and ATPase inhibition pattern by ADP and ATP/adenosine
5'-O-(thiotriphosphate) (ATP
S). hRAD51 fully saturates
with ATP/ATPS regardless of DNA cofactor (KD 
5 µM; 1 ATP/1 hRAD51). The binding of ADP to hRAD51
appeared bimodal. The first mode was identical to ATP/ATPS binding
(Kapp1 3 µM; 1 ADP/1 hRAD51),
while a second mode occurred at elevated ADP concentrations
(Kapp2 
125 µM; >1 ADP/1
hRAD51). We could detect ADP 
ATP exchange in the high affinity ADP
binding mode (Kapp1) but not the low affinity
binding mode (Kapp2). At low ATP concentrations
(<0.3 mM), ADP and ATPS competitively inhibit the
hRAD51 ATPase (Km(app) > Km). However, at high ATP (>0.3 mM),
the hRAD51 ATPase was stimulated by concentrations of ATPS that were
20-fold above the KD. Ammonium sulfate plus
spermidine decreased the affinity of hRAD51 for ADP substantially
(~10-fold) and ATP modestly (~3-fold). Our results suggest that ATP
binding is not rate-limiting but that the inability to sustain an
active nucleoprotein filament probably restricts the hRAD51 ATPase.
To whom correspondence and reprint requests should be
addressed: Kimmel Cancer Center, BLSB933, 233 S. 10th St., Rm.
933, Philadelphia, PA 19107. Tel.: 213-503-1346; Fax:
215-923-1098; E-mail: rfishel@hendrix.jci.tju.edu.
This article has been cited by other articles:
|
|
 |
|
  T. van der Heijden, R. Seidel, M. Modesti, R. Kanaar, C. Wyman, and C. Dekker Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules Nucleic Acids Res., September 27, 2007; 35(17): 5646 - 5657. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Li, X.-P. Zhang, J. A. Solinger, K. Kiianitsa, X. Yu, E. H. Egelman, and W.-D. Heyer Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics Nucleic Acids Res., June 22, 2007; (2007) gkm412v2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S. Shim, C. Schmutte, G. Tombline, C. D. Heinen, and R. Fishel hXRCC2 Enhances ADP/ATP Processing and Strand Exchange by hRAD51 J. Biol. Chem., July 16, 2004; 279(29): 30385 - 30394. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. V. Bugreev and A. V. Mazin Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity PNAS, July 6, 2004; 101(27): 9988 - 9993. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Trojanek, T. Ho, L. Del Valle, M. Nowicki, J. Y. Wang, A. Lassak, F. Peruzzi, K. Khalili, T. Skorski, and K. Reiss Role of the Insulin-Like Growth Factor I/Insulin Receptor Substrate 1 Axis in Rad51 Trafficking and DNA Repair by Homologous Recombination Mol. Cell. Biol., November 1, 2003; 23(21): 7510 - 7524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Tombline and R. Fishel Biochemical Characterization of the Human RAD51 Protein. I. ATP HYDROLYSIS J. Biol. Chem., April 19, 2002; 277(17): 14417 - 14425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Tombline, C. D. Heinen, K.-S. Shim, and R. Fishel Biochemical Characterization of the Human RAD51 Protein. III. MODULATION OF DNA BINDING BY ADENOSINE NUCLEOTIDES J. Biol. Chem., April 19, 2002; 277(17): 14434 - 14442. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
