4.7 Article

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

Journal

SCIENTIFIC REPORTS
Volume 13, Issue 1, Pages -

Publisher

NATURE PORTFOLIO
DOI: 10.1038/s41598-023-28564-6

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The N-terminal domain (NTD) of Tdp1, which is understudied and poorly conserved, plays a critical role in the removal of TOP1-DNA adducts in cells. It is proposed that the NTD serves as a regulatory domain, coordinating the stabilization of the DNA-adducted end and facilitating hydrolysis.
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.

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