# Supplement Request for a Fluorometer

> **NIH NIH R01** · SAINT LOUIS UNIVERSITY · 2020 · $71,307

## Abstract

PROJECT SUMMARY
 Homologous recombination (HR) is critical for the maintenance of genomic stability, and functions to
eliminate DNA double-strand breaks and chromosomal lesions. Mutations in proteins that coordinate HR result
in a variety of cancers and hereditary disorders. HR is initiated when a double-stranded DNA break is
nucleolytically resected to generate single-stranded DNA (ssDNA) overhangs, which are readily coated and
protected by Replication Protein A (RPA). RPA is then replaced by the RAD51 recombinase, which forms long
nucleoprotein filaments to catalyze downstream strand exchange reactions to promote HR. A host of pro- and
anti-HR mediator proteins control the assembly of the RAD51 nucleoprotein filament on RPA-coated ssDNA;
thereby dictating the temporal and spatial establishment of HR. Mutations/defects in mediator proteins such as
BRCA2 result in severe deregulation of HR causing genomic instability and associated cancers. As in many
metabolic processes associated with replication, repair and remodeling of DNA, multiple proteins with DNA
binding capacity function together on the same DNA template. Investigating the mechanism of how a single
enzyme functions in such a multi-protein milieu is technically challenging. Our approach to address this challenge
uses site-specific fluorescent versions of a protein of interest that produces a change in fluorescence upon
specifically binding to ssDNA. We have utilized non-canonical amino acids (ncAA) and strain-promoted
cycloaddition to generate fluorescent versions of RPA (RPAf) that accurately reports its dynamics on ssDNA,
enabling us to investigate how RAD51 nucleoprotein filaments are formed on RPA coated substrates. Using this
methodology, we have uncovered how each domain within RPA binds/dissociates on ssDNA and present a new
paradigm for RPA function. We will capitalize on this technological advancement to determine how RPA is
situated on DNA during HR and how its DNA binding domains undergo micro-dissociation during RPA-exchange
(Aim 1). We will decipher how pro-HR mediator proteins displace RPA while promoting the formation of RAD51
nucleoprotein filaments during HR (Aim 2). These aims will be achieved using ncAA-based approaches to
generate fluorescent proteins and a combination of ensemble and single molecule biophysical methodologies to
obtain kinetic parameters for the individual steps in their mechanism of action. Beyond providing mechanistic
insights on mechanism of HR, our studies will advance technology for investigating the dynamics of multi-protein
DNA reactions.

## Key facts

- **NIH application ID:** 10135255
- **Project number:** 3R01GM133967-02S1
- **Recipient organization:** SAINT LOUIS UNIVERSITY
- **Principal Investigator:** Edwin Antony
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $71,307
- **Award type:** 3
- **Project period:** 2019-09-10 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10135255

## Citation

> US National Institutes of Health, RePORTER application 10135255, Supplement Request for a Fluorometer (3R01GM133967-02S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10135255. Licensed CC0.

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