Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function

Peter Chi, Stephen Van Komen, Michael G. Sehorn, Stefan Sigurdsson, Patrick Sung

Research output: Contribution to journalArticlepeer-review

120 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)381-391
Number of pages11
JournalDNA Repair
Volume5
Issue number3
DOIs
StatePublished - Mar 7 2006
Externally publishedYes

Keywords

  • ATP hydrolysis
  • Human Rad51
  • Presynaptic filament
  • Walker A motif

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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