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.