Cancer predispositions in humans and severe DNA damage phenotypes in yeast result from defects in the Mre11-Rad50-Nbs1 (MRN) complex. MRN plays central and essential roles in repairing DNA double-strand breaks (DSBs) during homologous recombination repair as well as acting in meiosis, antibody hypermutation, telomere maintenance, and DNA damage signaling through ATM kinase. Yet, detailed mechanistic insights into these diverse MRN functions remain limited. The Mre11 nuclease complex with the Rad50 ATPase is conserved from archaea to humans and is regulated by Nbs1 in S. pombe and humans. We propose three Specific Aims to advance knowledge of MRN structural biochemistry, conformations, and interactions relevant to DNA damage repair and signaling functions. To accomplish these Aims, we will apply advanced biophysical techniques, including synchrotron solution X-ray scattering and atomic resolution crystal structure technologies in concert with genetic and mutational analyses in yeast. The proposed integrated biophysical and genetic studies will test hypotheses regarding Mre11's role in DNA target specificity and processing, Rad50's role in ATP-induced conformational controls and architectural interactions, and Nbs1's role in modulating Mre11 and Rad50 activities. The expected results will characterize functionally key Mre11, Rad50 and Nbs1 protein-protein and protein-DNA interfaces, conformations, and interaction architectures. Furthermore, the DNA damage sensitivity observed in the absence of any one member of the MRN complex suggests that our results will form a platform to test the utility of inhibitors that increase cellular sensitivity to ionizing radiation and other DNA damaging agents used for cancer radiotherapy and chemotherapy. Overall the results will connect MRN to cellular outcomes and human disease by defining interactions and mechanisms controlling genetic integrity, cancer resistance, radiotherapy resistance, and predispositions to cancer.

Public Health Relevance

Major goals for cancer research for the past decade have been the identification of the molecular underpinnings of cancer and the development of novel approaches to intervention. A major advance has been the characterization of the Mre11-Rad50-Nbs1 (MRN) complex as a critical suppressor of tumorigenesis and a resistance factor for current therapies. The proposed research will provide insights into the molecular mechanisms for MRN functions relevant to both cancer avoidance and interventions.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Radiation Therapeutics and Biology Study Section (RTB)
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Knowlton, John R
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Scripps Research Institute
La Jolla
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Syed, Aleem; Tainer, John A (2018) The MRE11-RAD50-NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair. Annu Rev Biochem 87:263-294
Limbo, Oliver; Yamada, Yoshiki; Russell, Paul (2018) Mre11-Rad50-dependent activity of ATM/Tel1 at DNA breaks and telomeres in the absence of Nbs1. Mol Biol Cell 29:1389-1399
Moiani, Davide; Ronato, Daryl A; Brosey, Chris A et al. (2018) Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities. Methods Enzymol 601:205-241
Reubens, Michael C; Rozenzhak, Sophie; Russell, Paul (2017) Multi-BRCT Domain Protein Brc1 Links Rhp18/Rad18 and ?H2A To Maintain Genome Stability during S Phase. Mol Cell Biol 37:
Sanchez, Arancha; Gadaleta, Mariana C; Limbo, Oliver et al. (2017) Lingering single-strand breaks trigger Rad51-independent homology-directed repair of collapsed replication forks in the polynucleotide kinase/phosphatase mutant of fission yeast. PLoS Genet 13:e1007013
Dutta, Arijit; Eckelmann, Bradley; Adhikari, Sanjay et al. (2017) Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex. Nucleic Acids Res 45:2585-2599
Guo, Lan; Ganguly, Abantika; Sun, Lingling et al. (2016) Global Fitness Profiling Identifies Arsenic and Cadmium Tolerance Mechanisms in Fission Yeast. G3 (Bethesda) 6:3317-3333
Jensen, Kristi L; Russell, Paul (2016) Ctp1-dependent clipping and resection of DNA double-strand breaks by Mre11 endonuclease complex are not genetically separable. Nucleic Acids Res 44:8241-9
Petersen, Janni; Russell, Paul (2016) Growth and the Environment of Schizosaccharomyces pombe. Cold Spring Harb Protoc 2016:pdb.top079764
Sánchez, Arancha; Russell, Paul (2015) Ku stabilizes replication forks in the absence of Brc1. PLoS One 10:e0126598

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