The goal of this application is to address critical gaps in our understanding of the Non-homologous end joining (NHEJ) DNA repair pathway using new single molecule methods. NHEJ is the main pathway for repair of DNA double strand breaks (DSBs), the most cytotoxic form of DNA damage, resulting from Ionizing Radiation (IR) and chemotherapeutics. Mutations in NHEJ proteins are associated with genomic instability, IR sensitivity and severe combined immunodeficiency (SCID). Consequently, the targeted inhibition of the NHEJ pathway is of importance for sensitization of cancer cells in IR therapy. The mechanisms that control NHEJ play a key role in development and in response to cancer therapy, but the current state of knowledge regarding the NHEJ repair process is limited. We especially know very little about the physical nature of the NHEJ complex and how it is assembled, as common biochemical, structural and cell biology methods are limited in their capacities to resolve this information. Without this level of understanding, insights into NHEJ mutations that cause IR sensitivity and immunodeficiency and strategies for inhibiting NHEJ in neoplastic cells remain stagnant. In this application we utilize innovative single-molecule methods to define the NHEJ repair process. We use an array of new single-molecule biochemical methods to define the two critical steps of NHEJ: Assembly of NHEJ complex on DNA ends (Aim-1) and synapsis of DNA ends (Aim-2). Within these aims we will determine the specific functional organization of NHEJ complex components, their dependence on DNA end chemistry, and the consequence of their clinical and structural mutations.
In Aim -3 we study NHEJ in cells. We will define the nanoscale architecture of NHEJ complexes in cells and determine their association with cellular DNA damage response (DDR) factors and characterize how these are modulated in different types of DSB lesions. Finally, we will determine the consequence of clinical and structural mutations in NHEJ proteins and the effect of NHEJ inhibitors on the organization of NHEJ complexes in cells. Our results will provide a platform for addressing novel hypotheses with cutting-edge single-molecule technology, with enormous potential for advancing the field of DNA damage research.

Public Health Relevance

The goal of this proposal is to establish the molecular mechanism of the non-homologous end joining (NHEJ) DNA double strand break (DSB) repair pathway, which is the primary DSB repair pathway in human cells, using innovative technology. DSBs disrepair causes genomic instability and cancer, while dysfunctional NHEJ is associated with severe human syndromes whose patients display features of cellular radiosensitivity and severe combined immunodeficiency. Furthering our understanding of the mechanism of NHEJ and related disease would bring us closer to mechanism-based therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM108119-04
Application #
9444510
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Willis, Kristine Amalee
Project Start
2015-03-01
Project End
2020-02-29
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
New York University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Whelan, Donna R; Lee, Wei Ting C; Yin, Yandong et al. (2018) Spatiotemporal dynamics of homologous recombination repair at single collapsed replication forks. Nat Commun 9:3882
Nemoz, Clement; Ropars, Virginie; Frit, Philippe et al. (2018) XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining. Nat Struct Mol Biol 25:971-980
D'Alessandro, Giuseppina; Whelan, Donna Rose; Howard, Sean Michael et al. (2018) BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment. Nat Commun 9:5376
Wang, John; Yin, Yandong; Lau, Stephanie et al. (2018) Anosmin1 Shuttles Fgf to Facilitate Its Diffusion, Increase Its Local Concentration, and Induce Sensory Organs. Dev Cell 46:751-766.e12
Tonzi, Peter; Yin, Yandong; Lee, Chelsea Wei Ting et al. (2018) Translesion polymerase kappa-dependent DNA synthesis underlies replication fork recovery. Elife 7:
Rona, Gergely; Roberti, Domenico; Yin, Yandong et al. (2018) PARP1-dependent recruitment of the FBXL10-RNF68-RNF2 ubiquitin ligase to sites of DNA damage controls H2A.Z loading. Elife 7:
Baranes-Bachar, Keren; Levy-Barda, Adva; Oehler, Judith et al. (2018) The Ubiquitin E3/E4 Ligase UBE4A Adjusts Protein Ubiquitylation and Accumulation at Sites of DNA Damage, Facilitating Double-Strand Break Repair. Mol Cell 69:866-878.e7
Bermudez-Hernandez, Keria; Keegan, Sarah; Whelan, Donna R et al. (2017) A Method for Quantifying Molecular Interactions Using Stochastic Modelling and Super-Resolution Microscopy. Sci Rep 7:14882
Reid, Dylan A; Conlin, Michael P; Yin, Yandong et al. (2017) Bridging of double-stranded breaks by the nonhomologous end-joining ligation complex is modulated by DNA end chemistry. Nucleic Acids Res 45:1872-1878
Conlin, Michael P; Reid, Dylan A; Small, George W et al. (2017) DNA Ligase IV Guides End-Processing Choice during Nonhomologous End Joining. Cell Rep 20:2810-2819

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