# Function and Evolutionary Origins of the RAG Endonuclease

> **NIH NIH R01** · YALE UNIVERSITY · 2023 · $624,777

## Abstract

SUMMARY
The RAG recombinase is a domesticated transposase that initiates V(D)J recombination and contributes
significantly to genome instability. To understand the mechanisms that protect the genome from dangerous
RAG endonuclease/transposase activity, we have taken a distinctive approach that melds evolutionary
biology with biochemistry and structural biology. From structures of ancestral RAG-like (RAGL) transposases,
we discovered RAG’s fundamental modular organization, an “on-off” switch that controls properly regulated
(“coupled”) cleavage, a novel DNA binding module that disrupts proper target site selection, and four
evolutionary adaptations in RAG that together provide powerful, multilayered protection against transposition.
These advances helped establish our current paradigm for RAG’s evolutionary origins and support a “DNA
confinement” model to explain errors in RAG targeting. Using these novel conceptual frameworks and our
recent discovery of a critical “missing link” in RAG’s evolutionary history, we will pursue our central objective:
to understand the mechanisms that ensure that RAG cuts appropriate targets in a properly orchestrated
(“coupled”) manner as well as the mechanisms that protect against catastrophic insertional mutagenesis due
to transposition into the genome. To achieve this objective, we will pursue the following aims:
Aim 1. Determine the evolutionary, structural, and biochemical basis of the RAGL→RAG transition.
We will systematically dissect the activity and structure of “missing link” RAGL transposases and rigorously
test the predictions of our DNA confinement and “on-off” switch models using in vitro protein biochemistry, a
suit of in vivo cleavage and transposition assays, cryo-electron microscopy, and chimeric RAG enzymes
engineered to possess carefully perturbed DNA binding and cleavage activities.
Aim 2. Determine the mechanisms by which RAG2 suppresses RAG-mediated transposition in vivo.
RAG2 and, surprisingly, “missing link” RAG2L proteins, possess an acidic hinge domain that powerfully
suppresses transposition, leading us to propose that RAG2L arose early in evolution as an “antitoxin” to
suppress the genotoxic potential of RAG1L (the transposase “toxin”). We will determine the protein residues
and mechanisms that mediate the suppressive activity of the acidic hinge and a second suppressive region
in RAG2, the LF2F3 loop, using an array of biochemical reconstitution and proximity labeling approaches.
Aim 3. Determine the biological and genomic consequences of hyperactivated/dysregulated RAG in
cells and mice. The goal of this aim is to connect mechanistic understanding to biological outcome. Using in
vivo transposition assays and mice harboring mutant RAG alleles, we will answer two outstanding questions:
i) Which RAG adaptations are needed to suppress RAG-mediated transposition from one site in the genome
to another? ii) What are the consequences for the genome, lymphoid development, and tumorigenesis, of
u...

## Key facts

- **NIH application ID:** 10801641
- **Project number:** 2R01AI137079-06
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** David G. Schatz
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $624,777
- **Award type:** 2
- **Project period:** 2018-09-11 → 2028-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10801641

## Citation

> US National Institutes of Health, RePORTER application 10801641, Function and Evolutionary Origins of the RAG Endonuclease (2R01AI137079-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10801641. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*