Long-Distance Lateral Diffusion of Human Rad51 on Double-Stranded DNA

Rad51 is the primary eukaryotic recombinase responsible for initiating DNA strand exchange during homologous recombination. Although the subject of intense study for over a decade, many molecular details of the reactions promoted by Rad51 and related recombinases remain unknown. Using total internal...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 103; no. 5; pp. 1221 - 1226
Main Authors Granéli, Annette, Yeykal, Caitlyn C., Robertson, Ragan B., Greene, Eric C.
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 31.01.2006
National Acad Sciences
Subjects
A form DNA
Adenosine triphosphatases
Adenosine Triphosphate - chemistry
Biochemistry
Biological Sciences
Biotinylation
Cysteine - chemistry
Deoxyribonucleic acid
Diffusion
DNA
DNA - chemistry
DNA Repair
Evolution, Molecular
Fluorescence
Fluorescence microscopy
Homologous recombination
Humans
Hydrodynamics
Hydrolysis
Lipid bilayers
Microscopy
Microscopy, Fluorescence - methods
Molecular biology
Molecules
Proteins
Rad51 Recombinase - chemistry
Rad51 Recombinase - metabolism
Recombination, Genetic
Spectroscopy, Fourier Transform Infrared
Time Factors
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Summary:Rad51 is the primary eukaryotic recombinase responsible for initiating DNA strand exchange during homologous recombination. Although the subject of intense study for over a decade, many molecular details of the reactions promoted by Rad51 and related recombinases remain unknown. Using total internal reflection fluorescence microscopy, we directly visualized the behavior of individual Rad51 complexes on double-stranded DNA (dsDNA) molecules suspended in an extended configuration above a lipid bilayer. Here we show that complexes of Rad51 can bind to and slide freely along the helical axis of dsDNA. Sliding is bidirectional, does not require ATP hydrolysis, and displays properties consistent with a 1D random walk driven solely by thermal diffusion. The proteins move freely on the DNA for long periods of time; however, sliding terminates and the proteins become immobile upon encountering the free end of a linear dsDNA molecule. This study provides previously uncharacterized insights into the behaviors of human Rad51, which may apply to other members of the RecA-like family of recombinases.
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To whom correspondence should be addressed. E-mail: ecg2108@columbia.edu.
Conflict of interest statement: No conflicts declared.
Abbreviations: dsDNA, double-stranded DNA; TIRFM, total internal reflection fluorescence microscopy.
Author contributions: E.C.G. designed research; A.G. and C.C.Y. performed research; E.C.G. contributed new reagents/analytic tools; A.G., R.B.R., and E.C.G. analyzed data; and E.C.G. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Edited by Kiyoshi Mizuuchi, National Institutes of Health, Bethesda, MD, and approved December 9, 2005
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0508366103