# Mechanism and Fidelity of RAG mediated DNA recombination

> **NIH NIH R01** · NEW YORK UNIVERSITY SCHOOL OF MEDICINE · 2022 · $533,862

## Abstract

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.

## Key facts

- **NIH application ID:** 10404048
- **Project number:** 5R01AI153040-03
- **Recipient organization:** NEW YORK UNIVERSITY SCHOOL OF MEDICINE
- **Principal Investigator:** Eli Rothenberg
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $533,862
- **Award type:** 5
- **Project period:** 2020-06-10 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10404048

## Citation

> US National Institutes of Health, RePORTER application 10404048, Mechanism and Fidelity of RAG mediated DNA recombination (5R01AI153040-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10404048. Licensed CC0.

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