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Principal Investigator: Eli Rothenberg
Organization: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
Fiscal Year: 2024
Award: $533,862
Funding agency: National Institute of Allergy and Infectious Diseases
V(D)J recombination lies at the heart of antigen receptor diversity and adaptive immunity. The RAG
complex (RAG), which includes RAG1, RAG2 and HMGB1, initiates this critical process by binding
recombination signal sequences (RSSs) and creating DNA double-stranded breaks (DSBs). The
resulting breaks are repaired via the non-homologous end-joining (NHEJ) pathway, the predominant
DSB repair mechanism in mammalian cells. Mutations in RAG or NHEJ proteins cause defects in V(D)J
recombination leading to joining errors, chromosomal deletions and translocations, and genome
instability. Defective V(D)J recombination is associated with a range of human disorders including
cancer, common immune deficiency (CID) and severe combined immunodeficiency (SCID), and
ionizing radiation (IR) sensitivity.
Despite much progress in the field, a particularly critical step of V(D)J recombination–the
transition from RAG-mediated DNA cleavage to NHEJ-mediated DNA repair–remains poorly
understood. Two particularly glaring gaps in our knowledge of this process are: 1) What are the steps
and RAG-NHEJ factor interactions that mediate this process? and 2) How are the RAG and NHEJ
complexes organized and regulated (dysregulated) in the “recombination centers” within which V(D)J
recombination takes place in vivo? Research into these questions has been hampered by limitations
inherent in traditional biochemical, structural, and cell biological approaches, limitations that can now be
overcome by high-resolution single molecule methods.
In this application, we propose to address these knowledge gaps by defining the molecular
mechanism of the RAG-NHEJ handoff process and how its dysfunction leads to aberrant V(D)J
recombination. To accomplish this, we will use of an array of innovative single-molecule techniques and
assays. The proposed studies are supported by key preliminary experiments including the application
of single-molecule assays to monitor the RAG-NHEJ handoff process in vitro in real-time, and utilization
of super-resolution imaging of recombination complexes during transactions of V(D)J recombination in
cells.
Terms: <AL-1><AL-1 Protein><Address><Affect><Amphoterin><Amphoterin Gene><Antigen Receptors><Assay><B blood cells><B cell><B cells><B-Cells><B-Lymphocytes><B-cell><Binding><Bioassay><Biochemical><Biological><Biological Assay><Cancers><Cell Body><Cell Line><Cell Nucleus><CellLine><Cells><Chromatin><Chromosomal Deletion><Chromosomal Protein, Nonhistone, HMG1><Chromosomal Protein, Nonhistone, HMG1 Gene><Chromosomal dislocation><Chromosomal translocation><Chromosome Deletion><Complex><DNA><DNA Damage><DNA Damage Repair><DNA Double Strand Break><DNA Injury><DNA Ligation><DNA Recombination><DNA Repair><Defect><Deoxyribonucleic Acid><Disease><Disorder><Double Strand Break Repair><Dysfunction><Ephrin-A5><Event><FM1 Gene Product><Functional disorder><Genetic Alteration><Genetic Change><Genetic Recombination><Genetic Translocation><Genetic defect><Genome Instability><Genomic Instability><Goals><HMG-1><HMG-1 Gene><HMG-1 Protein><HMG1><HMG1 Gene><HMG3><HMG3 Gene><HMGB1><HMGB1 Protein><HMGB1 gene><Heart><Heparin-Binding Protein p30><High Mobility Group Box Protein 1><High Mobility Group Protein 1><High Mobility Group Protein 1 Gene><High-Mobility Group (Nonhistone Chromosomal) Protein 1><High-Mobility Group (Nonhistone Chromosomal) Protein 1 Gene><High-Mobility Group Box 1><High-Mobility Group Box 1 Gene><Human><IgK><Immune system><Immunodeficiency Disorder><Immunodeficiency Syndrome><Immunoglobulin V(D)J Rearrangement><Immunologic Deficiency Syndromes><Immunological Deficiency Syndromes><In Vitro><Individual><Ionizing Electromagnetic Radiation><Ionizing radiation><Kinetics><Knowledge><LERK-7 Protein><Malignant Neoplasms><Malignant Tumor><Mammalian Cell><Mediating><Methods><Modern Man><Molecular><Molecular Interaction><Monitor><Mutation><NHEJ><Non-Homologous End Joining><Non-homologous DNA End Joining><Nonhistone Chromosomal Protein HGM1><Nonhistone Chromosomal Protein HGM1 Gene><Nonhomologous DNA End Joining><Nonhomologous End Joining><Nuclear><Nucleic Acid Regulator Regions><Nucleic Acid Regulatory Sequences><Nucleus><Partial Monosomy><Pathway interactions><Peptide Signal Sequences><Phase><Physiopathology><Process><Proteins><RAGS Protein><REK7 Ligand AL-1><Radiation Sensitivity><Radiation Tolerance><Radiation-Ionizing Total><Radiosensitivity><Reaction><Recombination><Regulation><Regulatory Regions><Repulsive Axon Guidance Signal Protein><Research><Resolution><SBP-1><SBP-1 Gene><Series><Severe Combined Immunodeficiency><Severe Combined Immunodeficiency Syndrome><Severe Combined Immunologic Deficiency><Signal Peptide><Signal Sequences><Site><Strains Cell Lines><Sulfoglucuronyl Carbohydrate Binding Protein><Sulfoglucuronyl Carbohydrate Binding Protein Gene><System><Techniques><Therapeutic><Time><Unscheduled DNA Synthesis><V(D)J Rearrangement><V(D)J Recombination><VDJ rearrangement><VDJ recombination><Visualization><adaptive immunity><biologic><chromosome dislocation><chromosome translocation><combined T and B cell inborn immunodeficiency><cultured cell line><experiment><experimental research><experimental study><experiments><genetic regulatory element><genome mutation><hypoimmunity><immune deficiency><immune deficiency disorder><immunodeficiency><in vivo><innovate><innovation><innovative><innovative technologies><insight><ionizing output><malignancy><mutant><nano meter scale><nano meter sized><nanometer scale><nanometer sized><nanoscale><neoplasm/cancer><pathophysiology><pathway><protein signal sequence><radio-sensitivity><radiosensitive><reconstitute><reconstitution><recruit><repair><repaired><resolutions><response><single molecule><spatiotemporal><superresolution imaging><virtual>