TY - JOUR
T1 - Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function
AU - Chi, Peter
AU - Van Komen, Stephen
AU - Sehorn, Michael G.
AU - Sigurdsson, Stefan
AU - Sung, Patrick
N1 - Funding Information:
This work was supported by US National Institutes of Health research grants RO1ES07061 and RO1CA110415 and postdoctoral fellowship F32 GM074529.
PY - 2006/3/7
Y1 - 2006/3/7
N2 - 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.
AB - 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.
KW - ATP hydrolysis
KW - Human Rad51
KW - Presynaptic filament
KW - Walker A motif
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U2 - 10.1016/j.dnarep.2005.11.005
DO - 10.1016/j.dnarep.2005.11.005
M3 - Article
C2 - 16388992
AN - SCOPUS:32644466860
VL - 5
SP - 381
EP - 391
JO - DNA Repair
JF - DNA Repair
SN - 1568-7864
IS - 3
ER -