Visualizing the Disassembly of S. cerevisiae Rad51 Nucleoprotein Filaments

Ragan B. Robertson, Dana N. Moses, Young Ho Kwon, Pamela Chan, Weixing Zhao, Peter Chi, Hannah Klein, Patrick Sung, Eric C. Greene

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into elongated nucleoprotein filaments on DNA. We have used total internal reflection fluorescence microscopy and a DNA curtain assay to investigate the dynamics of individual Saccharomyces cerevisiae Rad51 nucleoprotein filaments. For these experiments the DNA molecules were end-labeled with single fluorescent semiconducting nanocrystals. The assembly and disassembly of the Rad51 nucleoprotein filaments were visualized by tracking the location of the labeled DNA end in real time. Using this approach, we have analyzed yeast Rad51 under a variety of different reaction conditions to assess parameters that impact the stability of the nucleoprotein filament. We show that Rad51 readily dissociates from DNA in the presence of ADP or in the absence of nucleotide cofactor, but that free ATP in solution confers a fivefold increase in the stability of the nucleoprotein filaments. We also probe how protein dissociation is coupled to ATP binding and hydrolysis by examining the effects of ATP concentration, and by the use of the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido) triphosphate and ATPase active-site mutants. Finally, we demonstrate that the Rad51 gain-of-function mutant I345T dissociates from DNA with kinetics nearly identical to that of wild-type Rad51, but assembles 30% more rapidly. Together, these results provide a framework for studying the biochemical behaviors of S. cerevisiae Rad51 nucleoprotein filaments at the single-molecule level.

Original languageEnglish (US)
Pages (from-to)703-720
Number of pages18
JournalJournal of Molecular Biology
Volume388
Issue number4
DOIs
StatePublished - May 15 2009
Externally publishedYes

Keywords

  • DNA curtain
  • homologous recombination
  • nucleoprotein filament
  • Rad51
  • single molecule

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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