Published in Cell
The breast cancer susceptibility protein, BRCA2, is essential for recombinational DNA repair. BRCA2 delivers RAD51 to double-stranded DNA (dsDNA) breaks through interaction with eight conserved, approximately 35 amino acid motifs, the BRC repeats. Here we show that the solitary BRC4 promotes assembly of RAD51 onto single-stranded DNA (ssDNA), but n...
Published in Nature Protocols
In this protocol, we describe a procedure to generate DNA dumbbells -single molecules of DNA with a microscopic bead attached at each end-and techniques for manipulating individual DNA dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA dumbbells...
Published in Cell
RecBCD is a DNA helicase comprising two motor subunits, RecB and RecD. Recognition of the recombination hotspot, chi, causes RecBCD to pause and reduce translocation speed. To understand this control of translocation, we used single-molecule visualization to compare RecBCD to the RecBCD(K177Q) mutant with a defective RecD motor. RecBCD(K177Q) pause...
Published in G Protein Coupled Receptors - Structure
In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemble averaging. To better understand the behavior of proteins that bind to and/or translocate on DNA, we have developed instrumentation that uses optical trapping, microfluidic solution delivery, and fluorescent microscopy to visualize either individual p...
Published in Molecular Cell
Rad54 protein plays an important role in the recombinational repair of double-strand DNA (dsDNA) breaks. It is a dsDNA-dependent ATPase that belongs to the Swi2/Snf2 family of chromatin-remodeling proteins. Rad54 remodels (1) DNA structure, (2) chromatin structure, and (3) Rad51-dsDNA complexes. These abilities imply that Rad54 moves along DNA. Her...
Published in Journal of Biological Chemistry
The Saccharomyces cerevisiae Tid1 protein is important for the recombinational repair of double-stranded DNA breaks during meiosis. Tid1 is a member of Swi2/Snf2 family of chromatin remodeling proteins and shares homology with Rad54. Members of this family hydrolyze ATP and promote 1) chromatin remodeling, 2) DNA topology alterations, and 3) displa...
Published in Journal of Biological Chemistry
Fluorescent fusion proteins are exceedingly useful for monitoring protein localization in situ or visualizing protein behavior at the single molecule level. Unfortunately, some proteins are rendered inactive by the fusion. To circumvent this problem, we fused a hyperactive RecA protein (RecA803 protein) to monomeric red fluorescent protein (mRFP1) ...
Published in Proceedings of the National Academy of Sciences
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 ext...
Published in Nature
Escherichia coli RecA is essential for the repair of DNA double-strand breaks by homologous recombination. Repair requires the formation of a RecA nucleoprotein filament. Previous studies have indicated a mechanism of filament assembly whereby slow nucleation of RecA protein on DNA is followed by rapid growth. However, many aspects of this process ...
