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 camptothecins (CPTs) such as the FDA approved CPT derivatives topotecan and irinotecan. Tdp1 contains two HKD-motifs that provide 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. hTdp1H493R and the analogous yeast mutant (Tdp1H432R) enhances cell sensitivity to CPT. In addition, the toxicity induced by this mutant is caused by the formation of a more stable Tdp1-DNA covalent intermediate, a rare characteristic for a DNA repair enzyme. However, this His to Arg substitution induces a minor toxic phenotype compared to other substitutions, such as the His432 to Asn substitution, which induces a Top1 dependent cellular lethality. A band depletion assay suggests that in vivo/cell Tdp1His432Asn remains in complex with Top1 on the DNA, which was not observed in a biochemical in vitro assay.

Biochemical studies revealed that Tdp1 catalysis is independent of the N-terminal domain. Among Tdp1 proteins, the N-terminal domain is poorly conserved in sequence and size (∼80aa for yeast and 140aa for human Tdp1).

We investigated the role of the N-terminal domain for Tdp1 activity in the cell. The N-terminal truncated proteins showed similar cellular distribution as the full-length proteins. Interestingly, the N-terminal truncated proteins did not display the toxicity that was observed with the full-length Tdp1 mutant proteins. This suggests that the N-terminal domain is a critical determinate of Tdp1 cellular function.

Preliminary results from our human cell line model shows similar results implying that the function of the N-terminal domain is conserved among Tdp1 proteins although it is poorly conserved. Further studies are necessary to ensure that these constructs are properly distributed. Moreover, the N-terminal domain of hTdp1 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.

Citation Format: Selma M. Cuya, Keith C. Wanzeck, Evan Q. Comeaux, Robert C. van Waardenburg. N-terminal domain of Tyrosyl-DNA phosphodiesterase I (Tdp1) is critical for its cellular function. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3327. doi:10.1158/1538-7445.AM2013-3327