# Function and Evolutionary Origins of the RAG Endonuclease

> **NIH NIH R01** · YALE UNIVERSITY · 2020 · $525,311

## Abstract

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SUMMARY
The RAG1/RAG2 recombinase, which initiates V(D)J recombination, is a defining feature of jawed
vertebrate adaptive immunity and is thought to have evolved from a transposable element. Key aspects of
RAG biochemistry and in vivo regulation are not understood, leaving large gaps in our understanding of the
mechanisms by which RAG contributes to genome instability and the development of cancer. The Transib
and ProtoRAG transposons, which encode RAG-like transposases, provide an entirely new "toolbox" with
which to fill these gaps. In unpublished work, we have: i) determined the structure of ProtoRAG-DNA
complexes by cryo-electron microscopy (EM); ii) obtained crystals of Transib transposase that diffract x-rays
to ~3Å resolution; iii) identified a key component of the mechanism that directs coordinated (coupled) DNA
cleavage by RAG; and iv) discovered two mechanisms that suppress RAG-mediated transposition in vivo.
We will use these novel tools and findings to accomplish our central objective: to determine the
biochemical, structural, and regulatory mechanisms that have evolved to orchestrate RAG function
and to ascertain the biological consequences of failures of these mechanisms. Our proposal is
organized around three core questions. First, what mechanisms explain coupled cleavage by RAG and why
do those mechanisms break down? Second, how do the different modules within RAG work together to
determine activity? And third, what protects the genome from RAG-mediated transposition and what are
the consequences when those mechanisms fail? These questions are addressed in two interwoven aims:
Aim 1: Determine the underpinnings of DNA recognition and coupled cleavage by RAG and RAG-
family transposases. ProtoRAG transposase binds and cleaves DNA in a manner with striking similarities
to improperly regulated cleavage by RAG. Using novel RAG-ProtoRAG chimeric proteins, biochemistry,
single molecule biophysics, and cryo-EM and x-ray crystallography, we will determine how DNA binding
domains, DNA bending, complex stability, and conformational changes contribute to coordinated vs.
uncoordinated cleavage in synaptic complexes formed by RAG and RAG-like transposases.
Aim 2: Determine the regulation, targeting, and biological consequences of transposition into the
mammalian genome by RAG. Building on our discovery of RAG mutants that uncouple DNA cleavage or
activate transposition in vivo, we will use a suite of in vitro and in vivo transposition, cleavage, and high-
throughput sequencing assays in normal and DNA repair-deficient cells to quantitate and map transposition
mediated by intact and mutant RAG enzymes. In addition, we will generate and analyze RAG-mutant mice
with regulatory defects in DNA cleavage and transposition. Together, our results will reveal how DNA repair
factors and RAG catalytic and regulatory modules have evolved to protect genome stability and shield
developing lymphocytes from malignant transformation during the process of V(D...

## Key facts

- **NIH application ID:** 9990659
- **Project number:** 5R01AI137079-03
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** David G. Schatz
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $525,311
- **Award type:** 5
- **Project period:** 2018-09-11 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9990659

## Citation

> US National Institutes of Health, RePORTER application 9990659, Function and Evolutionary Origins of the RAG Endonuclease (5R01AI137079-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9990659. Licensed CC0.

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