# Protein Dynamics in Site-Specific DNA Recombination

> **NIH NIH R01** · OHIO STATE UNIVERSITY · 2020 · $104,850

## Abstract

Project Summary/Abstract
This goal of this proposal is to advance our understanding of the mechanisms by which tyrosine recombinases
recognize and assemble on their target DNA sequences and achieve coordinated pairwise cleavage of DNA
strands to reach a recombined product. The proposal is significant because tyrosine recombinases are widely
used genome editing tools, but their potential for improving human health is currently hindered by lack of
understanding of their mechanisms for site selection and for control over activity and recombination direction
(i.e., integration versus excision). Innovation in this proposal arises from the application of solution NMR to
address mechanistic knowledge gaps left unanswered by the many high resolution crystal structures of
tyrosine recombinases in tetrameric complexes with DNA. Those structures have provided crisp snapshots of
some of the important intermediates in the recombination pathway, but provide limited insight into the
intermediates that precede them, or into the mechanisms that interconvert them. Our approach combines
powerful protein- and DNA-engineering with sophisticated isotope labeling and NMR methods to characterize
dynamics that enable interconversion of key intermediates in site-specific DNA recombination, focusing on
those leading to site-specific assembly of the tetrameric synaptic complex, allosteric control over DNA
cleavage, and isomerization of the Holliday junction (HJ) intermediate.
To understand the conformational changes in both protein and DNA that accompany site selection, dimer
assembly, tetramer synapsis and protomer activation, we will use solution NMR spectroscopy to: (1) determine
the solution structure of Cre recombinase alone, and bound in pre-synapsed complexes with loxP DNA; (2)
determine the role of protein dynamics in activating Cre for DNA cleavage by measuring dynamics in Cre and
pre-synaptic complexes with DNA; (3) study how DNA intrinsic dynamics affects Cre recognition, synaptic
assembly, control over Cre activity, and direction of recombination; (4) characterize the dynamic and allosteric
communication pathways that enable isomerization of the central HJ for progression through the recombination
reaction. To enable the NMR experiments on large homo-oligomeric complexes, we will leverage an arsenal of
reagents and techniques for selective labeling of protein and DNA molecules, and for assembly of chimeric
Cre-DNA complexes; together with uniform deuteration and TROSY methods, the simplified NMR spectra will
facilitate resonance assignments and quantitative relaxation measurements. The proposed studies will
advance our understanding of the role of dynamics in DNA recombination, and in DNA binding and remodeling
enzymes in general. This knowledge could broadly impact biotechnology and its biomedical applications by
facilitating the design of Cre variants with defined DNA sequence specificity and improved efficiency, and
suggest new avenues for controlling its act...

## Key facts

- **NIH application ID:** 9883005
- **Project number:** 5R01GM122432-04
- **Recipient organization:** OHIO STATE UNIVERSITY
- **Principal Investigator:** MARK P. FOSTER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $104,850
- **Award type:** 5
- **Project period:** 2017-02-01 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9883005

## Citation

> US National Institutes of Health, RePORTER application 9883005, Protein Dynamics in Site-Specific DNA Recombination (5R01GM122432-04). Retrieved via AI Analytics 2026-06-01 from https://api.ai-analytics.org/grant/nih/9883005. Licensed CC0.

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