The broad objective of this proposal is to visualize and quantify the dynamic behavior of biological processes at the single-molecule level. We have developed new experimental strategies for studying protein-DNA interactions at the single-molecule level. We devised several approaches wherein we could easily manipulate an individual DNA molecule and expose it to a variety of different proteins, enzymes, and buffers. Using fluorescent detection, we could image individual proteins on DNA or detect their action on individual DNA molecules, typically in real-time. We propose to continue to use this approach to study unresolved aspects of recombinational DNA repair, chromatin remodeling, and DNA replication. We seek to continue to address unresolved questions regarding the assembly and disassembly of RecA/Rad51 nucleoprotein filaments, their control and function in homology-directed DNA repair. In addition, we propose to expand our interests and success in the area of chromatin remodeling by motor proteins. And finally, we have initiated studies on single-molecule imaging of DNA replication, and propose to address important issues that intersect with recombinational DNA repair.

Public Health Relevance

The broad objective of this proposal is to study unresolved aspects of recombinational DNA repair, chromatin remodeling, and DNA replication. These are fundamental biological processes that are necessary for maintenance of chromosomal integrity. The research in this proposal will provide basic information about the protein-protein and protein-DNA interactions that are essential to normal cellular function. These processes must be properly coordinated to ensure accurate transmission of genetic information. Understanding the molecular mechanism of these processes, and their integration, should shed light abnormal biological events that lead to mutation, chromosomal translocations, and genome instability. Consequently, a detailed molecular understanding of the intersection and integration of these key biological processes is important to human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM064745-12S1
Application #
9276464
Study Section
Program Officer
Lewis, Catherine D
Project Start
2002-07-15
Project End
2020-03-31
Budget Start
2016-04-05
Budget End
2017-03-31
Support Year
12
Fiscal Year
2016
Total Cost
$99,697
Indirect Cost
Name
University of California Davis
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
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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