N-terminal domain of tyrosyl-DNA phosphodiesterase I regulates topoisomerase I-induced toxicity in cells

Abstract

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.

SCAN1 mutant TDP1 blocks the repair of DSB induced by TOP1 activity during gene transcription and promotes genome reorganisations and cell death in quiescent cells

“…In this work, we also complemented TDP1 -/- cells with a different catalytically inactive mutant of TDP1(5, 11). We failed to obtain high levels of TDP1 H263A expression in TDP1 -/- cells, in agreement with previous reports showing an inherent toxicity of TDP1 H263A expression in yeast and human cells (35–37). However, taking advantage of our inducible complementation system we were able to test the effect of this mutant when expressed in quiescent RPE-1 cells.…”

“…In this work, we also complemented TDP1 -/cells with a different catalytically inactive mutant of TDP1 (5,11). We failed to obtain high levels of TDP1 H263A expression in TDP1 -/cells, in agreement with previous reports showing an inherent toxicity of TDP1 H263A expression in yeast and human cells (35)(36)(37).…”

Multifunctional small biomolecules as key building blocks in the development of hydrogel-based strain sensors

“…Furthermore, these nucleobases promote biocompatibility in hydrogels because of their inherent biocompatibility 257 and capacity to modulate cellular interactions through ligand binding. 269,270 Although incorporating DNA-inspired molecules into hydrogels has shown promising results, further research is required to optimize their use in different hydrogel compositions, investigate their long-term stability, and improve their mechanical toughness without compromising their adhesion properties.…”