Our genomic DNA is under constant assault from endogenous and exogenous sources, which needs to be resolved to maintain cellular homeostasis. The eukaryotic DNA repair enzyme Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the hydrolysis of phosphodiester bonds that covalently link adducts to DNA-ends. Tdp1 utilizes two catalytic histidines to resolve a growing list of DNA-adducts. These DNA-adducts can be divided into two groups: small adducts, including oxidized nucleotides, RNA, and non-canonical nucleoside analogs, and large adducts, such as (drug-stabilized) topoisomerase-DNA covalent complexes or failed Schiff base reactions as occur between PARP1 and DNA. Many Tdp1 substrates are generated by chemotherapeutics linking Tdp1 to cancer drug resistance, making a compelling argument to develop small molecules that target Tdp1 as potential novel therapeutic agents. Tdp1's unique catalytic cycle, which is centered on the formation of Tdp1-DNA covalent reaction intermediate, allows for two principally different targeting strategies: (1) catalytic inhibition of Tdp1 catalysis to prevent Tdp1-mediated repair of DNA-adducts that enhances the effectivity of chemotherapeutics; and (2) poisoning of Tdp1 by stabilization of the Tdp1-DNA covalent reaction intermediate, which would increase the half-life of a potentially toxic DNA-adduct by preventing its resolution, analogous to topoisomerase targeted poisons such as topotecan or etoposide. The catalytic Tdp1 mutant that forms the molecular basis of the autosomal recessive neurodegenerative disease spinocerebellar ataxia with axonal neuropathy best illustrates this concept; however, no small molecules have been reported for this strategy. Herein, we concisely discuss the development of Tdp1 catalytic inhibitors and their results.

Protein degradation in all domains of life requires ATPases that unfold and inject proteins into compartmentalized proteolytic chambers. Proteasomal ATPases in eukaryotes and archaea contain poorly understood N-terminally conserved coiled-coil domains. In this study, we engineer disulfide crosslinks in the coiled-coils of the archaeal proteasomal ATPase (PAN) and report that its three identical coiled-coil domains can adopt three different conformations: (1) in-register and zipped, (2) in-register and partially unzipped, and (3) out-of-register. This conformational heterogeneity conflicts with PAN’s symmetrical OB-coiled-coil crystal structure but resembles the conformational heterogeneity of the 26S proteasomal ATPases’ coiled-coils. Furthermore, we find that one coiled-coil can be conformationally constrained even while unfolding substrates, and conformational changes in two of the coiled-coils regulate PAN switching between resting and active states. This switching functionally mimics similar states proposed for the 26S proteasome from cryo-EM. These findings thus build a mechanistic framework to understand regulation of proteasome activity.

The conserved eukaryotic DNA repair enzyme Tyrosyl-DNA phosphodiesterase I (Tdp1) removes a diverse array of adducts from the end of DNA strand breaks. Tdp1 specifically catalyzes the hydrolysis of phosphodiester linked DNA-adducts. These DNA lesions range from damaged nucleotides to peptide-DNA adducts to protein-DNA covalent complexes and are products of endogenously or exogenously induced insults or simply failed reaction products. These adducts include DNA inserted ribonucleotides and non-conventional nucleotides, as well as covalent reaction intermediates of DNA topoisomerases with DNA and a Tdp1-DNA adduct in trans. This implies that Tdp1 plays a role in maintaining genome stability and cellular homeostasis. Dysregulation of Tdp1 protein levels or catalysis shifts the equilibrium to genome instability and is associated with driving human pathologies such as cancer and neurodegeneration. In this review, we highlight the function of the N-terminal domain of Tdp1. This domain is understudied, structurally unresolved, and the least conserved in amino acid sequence and length compared to the rest of the enzyme. However, over time it emerged that the N-terminal domain was post-translationally modified by, among others, phosphorylation, SUMOylation, and Ubiquitinoylation, which regulate Tdp1 protein interactions with other DNA repair associated proteins, cellular localization, and Tdp1 protein stability.

Tyrosyl-DNA phosphodiesterase I (Tdp1) hydrolyzes phosphodiester-linked adducts from both ends of DNA. This includes the topoisomerase I (TOP1)-DNA covalent reaction intermediate that is the target of the camptothecin class of chemotherapeutics. Tdp1 two-step catalysis is centered on the formation of a Tdp1-DNA covalent complex (Tdp1cc) using two catalytic histidines. Here, we examined the role of the understudied, structurally undefined, and poorly conserved N-terminal domain (NTD) of Tdp1 in context of full-length protein in its ability to remove TOP1cc in cells. Using toxic Tdp1 mutants, we observed that the NTD is critical for Tdp1’s ability to remove TOP1-DNA adducts in yeast. Full-length and N-terminal truncated Tdp1 mutants showed similar expression levels and cellular distribution yet an inversed TOP1-dependent toxicity. Single turnover catalysis was significantly different between full-length and truncated catalytic mutants but not wild-type enzyme, suggesting that Tdp1 mutants depend on the NTD for catalysis. These observations suggest that the NTD plays a critical role in the regulation of Tdp1 activity and interaction with protein-DNA adducts such as TOP1cc in cells. We propose that the NTD is a regulatory domain and coordinates stabilization of the DNA-adducted end within the catalytic pocket to access the phosphodiester linkage for hydrolysis.