The general goal is to identify and understand the molecular mechanisms of recombinational DNA repair, a key pathway for preserving genomic integrity. This application focuses on the Rad51 paralogs, a class of proteins that has been notoriously difficult to study, and whose mechanistic contribution to recombination remains to be defined. The Rad51 paralogs act at a pivotal point, namely the assembly and maintenance of the Rad51- ssDNA filament, an essential intermediate for high-fidelity template-directed repair by homologous recombination DNA. In this application, two principal investigators (Heyer and Kowalczykowski) combine their expertise in a comprehensive approach to advance our understanding of Rad51 paralog function in DNA repair in yeast and humans utilizing single-molecule biophysics, ensemble biochemistry, and yeast genetics.
The Specific Aims are: (1) Determine the functional specialization of yeast Rad51 paralogs in yRad51-ssDNA filament assembly. The presynaptic Rad51-ssDNA filament exists in a meta-stable balance between its assembly and disassembly. We will determine the functional specialization of the Rad51 paralogs in Rad51 filament formation at gaps versus DSB breaks and determine potential selectivity for specific anti-recombinases. (2) Determine functional consequences of human RAD51 paralog action. To ascertain the functions of the RAD51 paralogs, their biochemical behavior will be examined. We will determine their DNA binding specificity, their capacity to stimulate DNA strand exchange promoted by hRAD51, and their ability to stabilize hRAD51 nucleoprotein filaments from disassembly both intrinsically and by helicases (BLM, RECQL5, FANCJ, and FBH1);the mechanisms underlying any effect will be determined. We will also examine whether other proteins, which interact with the RAD51 paralogs, function to regulate the formation or stability of RAD51 filaments. (3) Use single-molecule analyses to determine how the human RAD51 paralogs affect the dynamic behavior of hRAD51-ssDNA filaments. Testing the paradigm established in the yeast studies, we will measure whether the human RAD51 paralogs alter the rate of hRAD51 filament assembly or disassembly. The paralogs could, in principle, increase the nucleation frequency or the filament growth rate, or they could decrease the dissociation rate. These measurements will use direct imaging by single-molecule fluorescence microscopy of hRAD51 filament dynamics on individual molecules of DNA.

Public Health Relevance

Many modalities of anti-tumor therapy induce directly or indirectly DNA double-stranded breaks or gaps, and homologous recombination is a central DNA damage repair pathway, whose primary function is to repair such DNA damage. The proposed work focuses on a poorly understood class of homologous recombination proteins, the RAD51 paralogs that function in the assembly of the RAD51 filament. The expected results will lead to an improved mechanistic understanding of recombinational DNA repair, which is essential for understanding how mutations in the paralogs contribute to cancer predisposition and fundamental for designing biological approaches to improve the efficacy of DNA damage-based anti-tumor therapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA154920-02
Application #
8338889
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Pelroy, Richard
Project Start
2011-09-26
Project End
2016-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
2
Fiscal Year
2012
Total Cost
$519,850
Indirect Cost
$182,285
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
Wright, William Douglass; Heyer, Wolf-Dietrich (2014) Rad54 functions as a heteroduplex DNA pump modulated by its DNA substrates and Rad51 during D loop formation. Mol Cell 53:420-32
Zinovyev, Andrei; Kuperstein, Inna; Barillot, Emmanuel et al. (2013) Synthetic lethality between gene defects affecting a single non-essential molecular pathway with reversible steps. PLoS Comput Biol 9:e1003016