# Mechanisms of DNA hand-off during lesion repair in BER and NER

> **NIH NIH R01** · SAINT LOUIS UNIVERSITY · 2021 · $318,150

## Abstract

Exposure to environmental toxins, radiation and errors in endogenous DNA metabolism give rise to DNA
damage. Knowledge of the cellular DNA repair mechanisms that correct such DNA lesions are vital towards
combating genomic instability – a prevailing cause of cancers and associated disorders. To correct such errors,
double stranded DNA is unwound and the transiently opened single-stranded DNA (ssDNA) is protected and
coated by Replication Protein A (RPA), a high affinity multi-domain enzyme. Formation of RPA-ssDNA
complexes trigger the DNA repair checkpoint response and is a key step in activating most DNA repair pathways.
ssDNA-bound by RPA is handed-off to lesion-specific DNA repair proteins. The precise mechanisms of how this
functional specificity is achieved is poorly resolved. Towards addressing this gap in knowledge, our long-term
goals are to answer the following questions: a) RPA physically interacts with over two dozen DNA processing
enzymes; how are these interactions determined and prioritized? b) RPA binds to ssDNA with high affinity (KD
>10-10 M); how do DNA metabolic enzymes that bind to DNA with micromolar affinities remove RPA? c) Does
RPA play a role in positioning the recruited enzymes (with appropriate polarity) onto the DNA? d) How are the
DNA and protein interaction activities of RPA tuned by post translational modifications? To address these
questions, and to investigate the dynamics of RPA in the presence of multiple other DNA binding enzymes, we
have successfully developed an experimental strategy where the individual DNA binding domains (DBDs) of
RPA are labeled with a fluorophore. Upon binding to ssDNA, a robust change in fluorescence is observed and
thus serves as a real-time reporter of its dynamics on DNA. We achieved this through incorporation of noncanonical
amino acids and attachment of fluorophores using strain promoted click chemistry. Using this
methodology, we have uncovered how each domain within RPA binds/dissociates on ssDNA and present a new
paradigm for RPA function. There are four DBDs (A, B, C and D) in RPA and, for over three decades, DBD-A &
B have been thought to bind with highest affinity based on biochemical investigation of isolated DBDs. These
findings have served as a foundation for all models of RPA in DNA replication, repair and recombination. Our
work capturing RPA dynamics in the full-length context reveals the opposite, where DBDs A & B are highly
dynamic whereas DBDs C & D are stable. These startling findings completely alter the existing paradigm for
RPA function and form the basis of the proposed work investigating how specific RPA interacting proteins (RIPs)
gain access to DNA. Specifically, RPA modeling by NEIL1 and UNG2 during base excision repair (Aim 1) and
by XPA during nucleotide excision repair (Aim 2) will be investigated. In addition, the role of phosphorylation in
determining RPA specificity in DNA repair will be explored (Aim 3). Results from the proposed work will delineate
h...

## Key facts

- **NIH application ID:** 10093089
- **Project number:** 5R01GM130746-04
- **Recipient organization:** SAINT LOUIS UNIVERSITY
- **Principal Investigator:** Edwin Antony
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $318,150
- **Award type:** 5
- **Project period:** 2019-02-06 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10093089

## Citation

> US National Institutes of Health, RePORTER application 10093089, Mechanisms of DNA hand-off during lesion repair in BER and NER (5R01GM130746-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10093089. Licensed CC0.

---

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