# Mechanisms of RPA, Recombinases, and Mediators in Homologous Recombination

> **NIH NIH R01** · SAINT LOUIS UNIVERSITY · 2020 · $303,000

## 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 HR mediators dictate the
pathological progression of many 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). The Breast Cancer Type 2 Susceptibility (BRCA2) protein
functions to remove and remodel RPA and promote binding of the RAD51 recombinase, which then catalyzes
strand exchange and drives recombination. Our long-term goals are to gain a mechanistic understanding of the
temporal sequence of ssDNA handoff between these HR mediators and how these mediators compete for
access to ssDNA and its overall contribution to diseases such as cancer. RPA is composed of multiple distinct
DNA binding domains (DBDs) and binds with high affinity to ssDNA. The intrinsic DNA binding dynamics (binding,
dissociation and remodeling) of individual DBDs are hypothesized to dictate when, where, and how RPA
functions. For several decades the four DBDs of RPA have be classified as high affinity (DBDs-A & B) and low
affinity (DBDs-C & D) based on experimental measurements of ssDNA binding affinity of isolated DBDs and
have shaped models for RPA mechanism in DNA replication, repair and recombination. Using non-canonical
amino acids, we developed a fluorescence-based method to capture the dynamics of individual DBDs in the
context of the full-length protein. In contrary to classical models, we uncovered that the high-affinity DBDs are
dynamic and are outcompeted by the trimerization core of RPA. In lieu of these exciting discoveries of RPA
mechanism, we here propose to uncover how the BRCA2 mediator influences the dynamics of RPA-DBDs to
promote the formation of the RAD51 filament during HR. We will establish how the context and type of DNA
structures that are encountered in HR affect RPA-DBD dynamics [Aim 1]. We will investigate the importance of
a regulatory hotspot in RPA that would modulate the interaction between RPA and the BRCA2-DSS1 complex
[Aim 2]. Finally, using fluorescent versions of BRCA2, RAD51 and RAD52, we will establish the mechanism of
ssDNA handoff from RPA to RAD51 during HR [Aim 3].

## Key facts

- **NIH application ID:** 10015322
- **Project number:** 5R01GM133967-02
- **Recipient organization:** SAINT LOUIS UNIVERSITY
- **Principal Investigator:** Edwin Antony
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $303,000
- **Award type:** 5
- **Project period:** 2019-09-10 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10015322

## Citation

> US National Institutes of Health, RePORTER application 10015322, Mechanisms of RPA, Recombinases, and Mediators in Homologous Recombination (5R01GM133967-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10015322. Licensed CC0.

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