Abstract 501: Tyrosyl-DNA phosphodiesterase I cellular function dependent on its N-terminal residues

Abstract

Abstract Tyrosyl-DNA phosphodiesterase I (Tdp1) is a highly conserved eukaryotic DNA repair enzyme that catalyzes the resolution of 3’ and 5’ phospho-DNA adducts. Tdp1 has been implicated in the repair of DNA topoisomerase I (Top1)-DNA covalent complexes reversibly stabilized by FDA approved camptothecins (CPTs) derivatives topotecan and irinotecan. Tdp1 activity relies on two catalytic histidines that function as a nucleophile and an acid-base residue. A mutation of the acid-base His to Arg (H493R) in human Tdp1 is associated with the rare recessive ataxia SCAN1. We defined alternative substitution of either catalytic histidine that induce cytotoxicity, reduce catalytic activity and enhances the requisite Tdp1-DNA covalent adduct lifetime in the cell. The phenotypes of the catalytic mutants provide excellent tools to study Tdp1 cellular function. Biochemical studies revealed that Tdp1 catalysis in vitro is independent of the N-terminal domain. Among Tdp1 proteins, the N-terminal domain is poorly conserved in sequence and size (79aa for yeast and 148aa for human Tdp1). Conversely, the N-terminal domain regulates the in vitro activity of these Tdp1 mutants. Additionally, we investigated the role of the N-terminal domain for Tdp1 activity in the yeast and human cell models. Expression of N-terminal truncated proteins showed similar cellular distribution as the full-length proteins. However, these N-terminal truncated Tdp1 mutants did not display the toxicity that was observed with the full-length Tdp1 mutant proteins. Our data suggests that the N-terminal domain is required to resolve protein-DNA covalent complexes, such as Top1. Indeed, preliminary results suggest that this domain is also critical to process Top2-DNA covalent complexes, but only in the presences of etoposide. We are currently investigating other protein-DNA adducts that are resolved by Tdp1. These results suggests that the N-terminal domain is a critical determinate of Tdp1 cellular function. However, further studies are necessary to ensure that these constructs are properly distributed and retain their catalytic activity. Additionally, the N-terminal domain of human Tdp1 is post-translational modified, while our preliminary results suggest that this domain is important for protein-protein interaction and Tdp1 recruitment to its substrates. Understanding Tdp1 substrate and protein-interactions are important in the development of Tdp1 as therapeutic target. This work is in part supported by the ADDA, UAB Cancer Comprehensive Center (P30CA013148), ACS-IRG-60-001-53, DOD OCRP (W81XWH-15-1-0198). Citation Format: Selma M. Cuya, Robert C.A.M. Van Waardenburg. Tyrosyl-DNA phosphodiesterase I cellular function dependent on its N-terminal residues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 501. doi:10.1158/1538-7445.AM2017-501