Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules

Jovencio Hilario, Ichiro Amitani, Ronald J. Baskin, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

95 Scopus citations

Abstract

Rad51 protein (Rad51) is central to recombinational repair of double-strand DNA breaks. It polymerizes onto DNA and promotes strand exchange between homologous chromosomes. We visualized the real-time assembly and disassembly of human Rad51 nucleoprotein filaments on double-stranded DNA by single-molecule fluorescence microscopy. Rad51 assembly extends the DNA by ≈65%. Nucleoprotein filament formation occurs via rapid nucleation followed by growth from these nuclei. Growth does not continue indefinitely, however, and nucleoprotein filaments terminate when ≈2 μm in length. The dependence of nascent filament formation on Rad51 concentration suggests that 2-3 Rad51 monomers are involved in nucleation. Rad51 nucleoprotein filaments are stable and remain extended when ATP hydrolysis is prevented; however, when permitted, filaments decrease in length as a result of conversion to ADP-bound nucleoprotein complexes and partial protein dissociation. Dissociation of Rad51 from dsDNA is slow and incomplete, thereby rationalizing the need for other proteins that facilitate disassembly.

Original languageEnglish (US)
Pages (from-to)361-368
Number of pages8
JournalProceedings of the National Academy of Sciences of the United States of America
Volume106
Issue number2
DOIs
StatePublished - Jan 13 2009

Keywords

  • Nucleation
  • RecA protein
  • Recombination
  • Self-assembly
  • Single-molecule

ASJC Scopus subject areas

  • General

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