Ionizing radiation and chemical agents induce strand breaks in DNA, which give rise to mutations and chromosomal alterations that can cause cancer. One of the chief biological defenses against DNA strand breaks is Double-Strand Break (DSB) repair, a complicated family of biologic pathways that repairs DNA strand breaks. Some modes of DSB Repair can proceed with full restoration of the DNA sequence and no resulting loss of genetic information, but others result in significant loss. The molecular underpinnings of DSB repair are a matter of intense study, but questions remain regarding biochemical requirements and pathway selection, given that they are not all equally effective in repairing chromosomes without loss of genetic information. Within these broader issues are more specific questions regarding the mechanism of recruitment of proteins that participate in some DSB repair pathways but not others. In baker's yeast (S. cerevisiae), Saw1 protein recruits the Rad1-Rad10 protein complex to DSB sites, probably by binding to Rad52 protein, but the biochemical details regarding how this occurs are not known in detail. This project will detail the molecular basis for recruitment of the Saw1, Rad10 and Rad52 proteins to sites of DSB repair in the yeast S. cerevisiae.
The specific aims of this proposal are to determine in an evolutionarily-conserved eukaryotic model system, the yeast, S. cerevisiae: 1) whether patterns of Saw1 and Rad10 recruitment to DSBs are altered depending on the length of the nonhomologous DNA sequence flanking the DSB site, 2) minimum binding domains of Rad52 and Saw1 required to form a Rad52-Saw1 complex, 3) whether human Rad52 can be recruited to yeast DSB sites in the absence of yeast Rad52 and 4) whether human Rad52 can recruit Saw1 to yeast DSB sites, and whether mutations that result in loss of the "strand annealing" function of Rad52 affect recruitment of Saw1.
These aims will be investigated primarily by innovative fluorescence microscopy experiments in which DSBs will be induced specifically at fluorescently labeled loci on different chromosomes in live yeast cells, and their repair monitored by fluorescence imaging of convergent fluorescent signals. Localization of fluorescently labeled Rad52, Saw1 and Rad10 to the DSBs will be used to study the effects on recruitment of varying the lengths of nonhomologous DNA sequences flanking the DSBs as well as mutations in the RAD52 and SAW1 genes. In vitro techniques will be used to further study important interactions between the Rad52 and Saw1 proteins. These experiments will address important questions regarding the biochemical requirements for recruitment of Rad52, Saw1 and Rad10 to DSB sites. Understanding the molecular basis for cancer and aging is important for advancing clinical strategies to minimize human suffering. A more detailed understanding of the mechanisms by which cells repair DNA will aid in finding new drug targets for pharmaceuticals that might minimize cancer risk and the ailments associated with aging.

Public Health Relevance

The proposed research will increase our understanding of the mechanism of regulation of a complex biological pathway that protects against cancer and aging known as Double-strand Break Repair. Results of this work may aid in developing cancer prevention strategies and methods to reduce suffering in the elderly by preventing some of the symptoms of aging.

Agency
National Institute of Health (NIH)
Type
Research Continuance Award (SC3)
Project #
2SC3GM093858-05
Application #
8742702
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Rogers, Michael E
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
California State University Northridge
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Northridge
State
CA
Country
United States
Zip Code
91330
Diamante, Graciel; Phan, Claire; Celis, Angie S et al. (2014) SAW1 is required for SDSA double-strand break repair in S. cerevisiae. Biochem Biophys Res Commun 445:602-7