Journal
DNA REPAIR
Volume 5, Issue 3, Pages 381-391Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.dnarep.2005.11.005
Keywords
human Rad51; presynaptic filament; Walker A motif; ATP hydrolysis
Categories
Funding
- NCI NIH HHS [R01CA110415] Funding Source: Medline
- NIEHS NIH HHS [R01ES07061] Funding Source: Medline
- NIGMS NIH HHS [F32 GM074529, F32 GM074529-01] Funding Source: Medline
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The Rad51 recombinase polymerizes on ssDNA to yield a right-handed nucleoprotein filament, called the presynaptic filament, that can search for homology in duplex DNA and pair the recombining DNA molecules to form a DNA joint. ATP is needed for presynaptic filament assembly and homologous DNA pairing, but the roles of ATP binding and ATP hydrolysis in the overall reaction scheme have not yet been clearly defined. To address this issue, we have constructed two mutants of hRad51, hRad51 K133A and hRad51 K133R, expressed these mutant variants in Escherichia coli, and purified them to near homogeneity. Both hRad51 mutant variants are greatly attenuated for ATPase activity, but hRad51 K133R retains the ability to protect DNA from restriction enzyme digest and induce topological changes in duplex DNA in an ATP-dependent manner, whereas the hRad51 K133A variant is inactive. With biochemical means, we show that the presynaptic filament becomes greatly stabilized when ATP hydrolysis is prevented, leading to an enhanced ability of the presynaptic filament to catalyze homologous pairing. These results help form the basis for understanding the functions of ATP binding and ATP hydrolysis in hRad51-mediated recombination reactions. (c) 2005 Elsevier B.V. All rights reserved.
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