Tyrosyl-DNA Phosphodiesterase I N-Terminal Domain Modifications and Interactions Regulate Cellular Function

Abstract

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.

Keywords: DNA metabolism; DNA topoisomerases; DNA-adducts; Tdp1; catalytic mechanism; post-translational modifications; protein–protein interactions.

Figure 1

Tyrosyl-DNA phosphodiesterase I (Tdp1) interaction and catalysis of adducted DNA. (A) Linear representation of human Tdp1 domain structure. The structurally unresolved N-terminal domain (amino acid 1–149) in gray and the structurally resolved catalytic domain (amino acid 150–608) in brown. Boxes show the location of the two nuclear localization signal (nls) and the two HKN-catalytic motifs with their residue numbers above. Post-translationally modified residues (black line) are shown: S81 is phosphorylated; K111 is SUMOylated; R361 and R586 are demethylated. (B) Carbon-backbone overlay in a cartoon representation of the resolved catalytic core domain structures of Human Tdp1 (blue, PDB:1NOP) [31] and yeast Tdp1 (gray, PDB:1Q32) [9]. Although the amino acid sequence homology is only 38% conserved (identical + similar residues), the structural conservation is remarkably high, as their catalytic residues are nearly superimposable (zoom), with some deviations occurring only at the periphery. (C) Surface electrostatic potential (estimation generated by MacPyMol) of human Tdp1. The ssDNA (green with DNA-phosphate backbone in orange) located in the positively charged DNA binding-gorge is covalently bonded to vanadate (yellow). The vanadate is also bond to the active site tyrosine of hTopo1 as part of the protease resistance hTopo1 peptide fragment positioned in the protein docking basin. This basin displays a more neutral charged bottom with neutral/negatively charged enclosed walls. In the red ellipse is the location where the non-adducted DNA strand interacts as revealed in the crystal structure by Flett et al. [32]. Electrostatic charge is represented in gradients from blue (positive) to white (neutral) to red (negative). (D) Tdp1 two step catalytic cycle represented from a structural perspective (adjusted from 1NOP structure shown above). The first HKN-motif provides Lys265 and Asn283 that stabilize the adducted phosphate group via formation of hydrogen-bonds, which is supported by hydrogen-bonds formed by Lys595 and Asn516 from the second HKN-motif. Lys595 and Asn 516 also function in an electron-relay mechanism to first provide a proton to the general acid base His493 (His^gab) that maintains the nucleophilic His263 (His^nuc) in its deprotonated phase [9,28,31,33]. After docking and stabilizing the adducted DNA strand, the nucleophilic His263 will hydrolyze the 3’phospho–tyrosyl bond by forming a 3’phospho–hystidyl linkage, covalently attaching Tdp1 to the end of the DNA (Step 1). This step releases Topo1 from the DNA after the general acid/base His493 donates its proton to the phenoxy anion of tyrosine to prevent reformation of the original DNA-adduct [34]. The now nucleophilic general acid/base His493 will activate a water molecule by accepting its proton, while the remaining hydroxyl will hydrolyze the Tdp1–DNA bond releasing Tdp1 from the DNA end. All structures were generated using MacPyMol (Molecular Graphics System, Schrödinger, LLC).