Mechanism of repair of 5′-topoisomerase II–DNA adducts by mammalian tyrosyl-DNA phosphodiesterase 2

Abstract

The Topoisomerase II (topo II) DNA incision and ligation cycle can be poisoned (e.g following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5′-phosphotyrosyl (5′-Y) linked topo II-DNA adducts. Here, X-ray structures of mouse Tdp2-DNA complexes reveal that a Tdp2 β-2-helix-β DNA damage binding “grasp”, helical “cap”, and DNA lesion binding elements fuse to form an elongated protein-DNA conjugate substrate interaction groove. The Tdp2 DNA binding surface is highly tailored for engagement of 5′-adducted ssDNA ends, and restricts non-specific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analyses support a single-metal ion catalytic mechanism for the endonuclease–exonuclease–phosphatase (EEP) nuclease superfamily, and establish a molecular framework for targeted small molecule blockade of Tdp2-mediated resistance to anti-cancer topoisomerase drugs.

Proteolytic Degradation of Topoisomerase II (Top2) Enables the Processing of Top2·DNA and Top2·RNA Covalent Complexes by Tyrosyl-DNA-Phosphodiesterase 2 (TDP2)

“…5F). These results are consistent with the crystal structures for TDP2, showing the enzyme has a narrow DNA binding pocket, optimal for accessing single-stranded DNA ends (26,27).…”

“…TDP2-RNA⅐DNA Complex Crystallization-Expression of the mouse TDP2 catalytic domain (mTdp cat residues 118-370) for crystallization was carried out as described (26). The TDP2-RNA⅐DNA complex was crystallized using a dual one nucleotide 5Ј-overhang crystallization strategy (26) and a 5Ј-phosphorylated self-annealing oligonucleotide.…”

“…a digoxigenin) is attached to the tyrosine, revealing the potential of TDP2 to hydrolyze tyrosyl-DNA bonds even when a peptide is attached to the tyrosine (25). Yet crystal structures show that TDP2 has a relatively tight catalytic site (26,27), suggesting Top2 might need to be proteolyzed or denatured for TDP2 to access the tyrosyl-DNA bond. Accordingly, cellular studies indicate that the 26 S proteasome is involved in the degradation of stabilized Top2cc (28,29).…”

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

“…2B). The preference of TDP2 for 5′ phosphotyrosyl bonds is consistent with recent X-ray data (27)(28)(29), but TDP2 also is active on 3′-substrates (20,26,30). Although TDP2 likely is the only enzyme of its kind (30), complete knockout of TDP2 in chicken DT40 cells and in mice was not lethal (20), possibly reflecting the power of redundancy in DNA repair.…”

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

“…It has also been reported that the multifunctional enzyme Tdp2 (also known as TTRAP or EAPII) is more efficient in catalyzing the resolution of 5Ј-phosphotyrosyl adducts than Tdp1 (39). However, Tdp2 is a member of the exonuclease-endonucleasephosphatase (or EEP) nuclease family that uses single Mg 2ϩ -dependent catalysis to cleave the 5-phosphotyrosine bond and functions in the nonhomologous end joining pathway (40,41). Thus, Tdp1 and Tdp2 constitute complementary DNA repair activities, with distinct reaction mechanisms (27,42,43).…”