# Reversal of residue-specific ADP-ribosylations by ADP-ribosyl-acceptor hydrolases

> **NIH NIH R01** · UNIVERSITY OF CINCINNATI · 2021 · $334,910

## Abstract

Abstract
Residue-specific, reversible protein ADP-ribosylations regulate a broad range of biological processes, including
DNA damage responses, gene expression, and cell death. Therefore, homeostasis of cellular ADP-
ribosylations is essential for maintaining genomic integrity. ADP-ribosyl-acceptor hydrolases (ARHs) are a
family of metalloenzymes that regulate site-specific ADP-ribosylations. ARH3 specifically reverses poly(ADP-
ribose) and mono-ADP-ribosylation at serine, a major site for modification following DNA damage, whereas
ARH1 cleaves mono-ADP-ribosylation at arginine. However, there is a fundamental gap in understanding of
substrate selectivity, catalysis, and function of specific activities of ARHs, which is largely due to the lack of
quantitative and convenient tools and insufficient structural information on substrate-bound active forms. The
objective of this application is to develop novel quantitative assays that selectively measure the reversal of
residue-specific ADP-ribosylations and to elucidate the mechanism of substrate selectivity and the role of each
enzymatic activity of ARH3. In support of this objective, we have developed a highly sensitive, quantitative, and
convenient fluorescence-based assays that specifically monitor the reversal of poly(ADP-ribose) or serine
mono-ADP-ribosylation by ARH3. We have also determined initial structures of ARH3 bound to intact
substrates. Guided by these strong preliminary data, we will pursue three specific aims to test our hypothesis
that the metal-coordination states and unique structural plasticity of ARHs are linked to substrate selectivity
and efficient catalysis. In Aim 1, we will fully develop quantitative fluorescence-based assays that selectively
monitor the reversal of residue-specific mono-ADP-ribosylations. In Aim 2, we will determine the structural
bases for the specific substrate recognition and cleavage by ARHs. In Aim 3, we will identify ARH3 separation-
of-function mutants to define the role of each enzymatic activity of ARH3. Our studies will provide new
quantitative tools to study diverse ADP-ribosylation-metabolizing enzymes in nature and advance our
understanding of the mechanisms and functions of ARHs as essential processes for maintaining life.

## Key facts

- **NIH application ID:** 10183062
- **Project number:** 1R01GM141226-01
- **Recipient organization:** UNIVERSITY OF CINCINNATI
- **Principal Investigator:** In-Kwon Kim
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $334,910
- **Award type:** 1
- **Project period:** 2021-05-01 → 2025-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10183062

## Citation

> US National Institutes of Health, RePORTER application 10183062, Reversal of residue-specific ADP-ribosylations by ADP-ribosyl-acceptor hydrolases (1R01GM141226-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10183062. Licensed CC0.

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