The long-term objective of this proposal is to understand the behavior of proteins at the level of solitary protein-DNA complexes. This proposal takes advantage of a novel single-molecule approach that can visualize the behavior and dynamic properties of individual complexes of proteins with DNA. Several different protein-DNA complexes will be examined;each is an essential component of the DNA recombination process. The proteins that will be examined include the DNA strand exchange proteins, RecA and Rad51;the mediators and modulators of RecA/Rad51 function;and the finally the nucleoprotein- and chromatin-remodeling translocase, Rad54 protein.
The specific aims are to: 1. Visualize and measure the assembly, disassembly, and polarity of RecA and Rad51 nucleoprotein filaments.
This aim addresses the core behavior of the RecA/Rad51 nucleoprotein filament - its dynamic behavior and its ability to find DNA homology. 2. Determine how the dynamic behavior of RecA/Rad51 nucleoprotein filaments is modified by competitors and mediators.
This aim addresses the question of how competitor, mediator, and motor proteins modulate the behavior of RecA and Rad51 filaments. 3. Define the role of translocation along dsDNA by Rad54 protein.
This aim addresses the functions of Rad54 translocation capacity and its ability to remodel protein-DNA complexes. The visualization of these proteins acting at the single-molecule level, in real-time, affords a completely new window into the behavior and function of these proteins. Each of these proteins is involved in the repair of DNA breaks by recombination, a process whose mechanism is not fully understood. Recently, new methods of visualizing the action of these repair enzymes on single-molecules of DNA have been developed. These methods can provide an unprecedented level of understanding of these intricate processes. These single-molecule methods will be used to define some of the molecular events comprising increasingly complicated biochemical processes that underpin recombinational DNA repair.
|Graham, James E; Marians, Kenneth J; Kowalczykowski, Stephen C (2017) Independent and Stochastic Action of DNA Polymerases in the Replisome. Cell 169:1201-1213.e17|
|Bell, Jason C; Kowalczykowski, Stephen C (2016) RecA: Regulation and Mechanism of a Molecular Search Engine. Trends Biochem Sci 41:491-507|
|Pavankumar, T L; Exell, J C; Kowalczykowski, S C (2016) Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases. Methods Enzymol 581:1-32|
|Bell, Jason C; Liu, Bian; Kowalczykowski, Stephen C (2015) Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function. Elife 4:e08646|
|Kowalczykowski, Stephen C (2015) An Overview of the Molecular Mechanisms of Recombinational DNA Repair. Cold Spring Harb Perspect Biol 7:|
|Liu, Bian; Baskin, Ronald J; Kowalczykowski, Stephen C (2013) DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate. Nature 500:482-5|
|Forget, Anthony L; Dombrowski, Christopher C; Amitani, Ichiro et al. (2013) Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy. Nat Protoc 8:525-38|
|Bell, Jason C; Plank, Jody L; Dombrowski, Christopher C et al. (2012) Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA. Nature 491:274-8|
|Forget, Anthony L; Kowalczykowski, Stephen C (2012) Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search. Nature 482:423-7|
|Forget, Anthony L; Kowalczykowski, Stephen C (2010) Single-molecule imaging brings Rad51 nucleoprotein filaments into focus. Trends Cell Biol 20:269-76|
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