# Mechanism and Targeting of V(D)J Recombination

> **NIH NIH R01** · YALE UNIVERSITY · 2020 · $465,511

## Abstract

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SUMMARY
Errors made by the RAG1/RAG2 endonuclease during V(D)J recombination can lead to genome instability
and the development of leukemia and lymphoma. One important cause of such instability is improper action
of RAG at "cryptic" recombination signal sequences (RSSs) that are present abundantly in the genome.
Our prior work provided mechanistic and structural insights into RAG protein-DNA complexes and revealed
that RAG1 and RAG2 bind to numerous sites in the genome outside of the antigen receptor loci, virtually all
of which are active promoters or enhancers. We further demonstrated that such "off-target" RAG binding
occurs through two modes, one driven by a histone code "reader" function of RAG2 (largely at promoters)
and the other dependent on regulatory portions of RAG1 (largely at enhancers). The thousands of off-target
RAG binding sites and the millions of cryptic RSSs in the genome raise fundamental questions about how
the genome is protected from devastating instability caused by RAG. The central objective of our
proposed experiments is to determine the rules that govern RAG off-target activity by
understanding the interactions that dictate RAG localization in the genome and the mechanisms
that determine which cryptic RSSs are cleaved by RAG and which are spared. We will use
complementary biochemical, biophysical, genetic, and genomic approaches to achieve the following aims:
Aim 1. Determine the rules governing the selection of cryptic RSS targets by RAG. We and others
have identified intriguing sequence and topological features of the cryptic RSSs cleaved by RAG, but it is
unknown to what extent, or how, these features dictate RAG activity. We will systematically determine how
cryptic RSS orientation, location, and sequence influence RAG off-target activity and in the process, test a
provocative new hypothesis that RAG acquires its targets in part through linear "tracking" along DNA.
Aim 2. Determine the domains and interactions directing RAG1 to active enhancers. Much off-target
cutting by RAG occurs in enhancers, but the molecular interactions that dictate localization of RAG to these
regions are not known. We will identify the portions of RAG1 and the RAG1-binding partners that specify
enhancer binding and test the hypothesis that RAG1, like RAG2, is a reader of the histone code.
Aim 3. Determine how retargeting of RAG alters cRSS selection and off-target cleavage patterns.
We will use RAG mutants and a novel, inducible in vivo targeting system to direct RAG to new sites in the
genome and determine the spectrum of new cleavage sites that arise. We will also reconstitute RAG-
mediated cleavage at "strong" cryptic RSSs normally ignored by RAG to uncover the epigenetic and
chromatin architectural parameters that specify RAG off-target activity.
 Together, our proposed studies have a dual significance, both for basic mechanisms of RAG function
and for the causes of cancer.

## Key facts

- **NIH application ID:** 9986608
- **Project number:** 5R01AI032524-29
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** David G. Schatz
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $465,511
- **Award type:** 5
- **Project period:** 1992-04-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9986608

## Citation

> US National Institutes of Health, RePORTER application 9986608, Mechanism and Targeting of V(D)J Recombination (5R01AI032524-29). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9986608. Licensed CC0.

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