# Structure and Specificity of Restriction-Modification (R-M) Systems

> **NIH NIH R35** · ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI · 2021 · $258,384

## Abstract

Restriction-modification (R-M) systems comprise the innate immune system in bacteria and archaea. Their
discovery ~50 years ago by Arber, Nathans, and Smith (1978 Nobel Prize in Physiology & Medicine)
opened the doors of modern biotechnology. Without R-M enzymes there would haven been no recombinant
DNA revolution and no gene technology, as we know it today. R-M systems range from simple Type II
enzymes to more complex families of enzymes that require ATP (Type I and III) or that encode both
endonuclease and methylation activities within the same polypeptide (Type IIL). Much has been learned
over the past two decades about the structure and mechanism of the simple Type II enzymes (such as
BamHI and FokI), providing fundamental insights into the basis of extreme protein-DNA selectivity and
lending to the creation of novel chimeric nucleases. However, much remains to be learned about the other
more complex families of R-M enzymes. EcoP15I is a prototype of the Type III R-M family that functions as
a pseudo-helicase or a molecular switch to communicate between distant DNA sites. The DNA is cleaved
when two EcoP15I complexes collide. Although Ecop15I was discovered >40 years ago there had been no
structural information. We have resolved the crystal structure of the complete Ecop15I complex. We will
carry out additional structural and functional studies aimed at understanding its mechanism of translocation
and DNA cleavage. MmeI is a prototype of the Type IIL R-M family that provides a natural platform for
engineering new DNA-binding specificities. Some success has already been achieved in this direction. We
will use structural information on MmeI-like enzymes to identify specificity determinants, which can then be
rationally mutated to generate new nucleases. We also look to understand how these enzymes control their
nuclease activity, as a means to prevent self-restriction while at the same time allowing for restriction of viral
DNA. Overall, we will uncover new structural principles by which these complex R-M systems communicate
and cleave DNA over long distances and how specificity determinants can be molded to create new
enzymes.

## Key facts

- **NIH application ID:** 10241952
- **Project number:** 5R35GM131780-03
- **Recipient organization:** ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
- **Principal Investigator:** ANEEL K. AGGARWAL
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $258,384
- **Award type:** 5
- **Project period:** 2019-09-01 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10241952

## Citation

> US National Institutes of Health, RePORTER application 10241952, Structure and Specificity of Restriction-Modification (R-M) Systems (5R35GM131780-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10241952. Licensed CC0.

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