Tyrosyl-DNA phosphodiesterase (Tdp1) participates in the repair of Top2-mediated DNA damage

Abstract

Agents targeting topoisomerases are active against a wide range of human tumors. Stabilization of covalent complexes, converting topoisomerases into DNA-damaging agents, is an essential aspect of cell killing by these drugs. A unique aspect of the repair of topoisomerase-mediated DNA damage is the requirement for pathways that can remove protein covalently bound to DNA. Tyrosyl-DNA phosphodiesterase (Tdp1) is an enzyme that removes phosphotyrosyl moieties bound to the 3' end of DNA. Cells lacking Tdp1 are hypersensitive to camptothecin, consistent with a role for Tdp1 in processing 3' phosphotyrosyl protein-DNA covalent complexes. Because Top2p forms a 5' phosphotyrosyl linkage with DNA, previous work predicted that Tdp1p would not be active against lesions involving Top2p. We found that deletion of the TDP1 gene in yeast confers hypersensitivity to Top2 targeting agents. Combining tdp1 mutations with deletions of genes involved in nonhomologous end joining, excision repair, or postreplication repair enhanced sensitivity to Top2 targeting drugs over the level seen with single mutants, suggesting that Tdp1 may function in collaboration with multiple pathways involved in strand break repair. tdp1 mutations can sensitize yeast cells to drugs targeting Top2 even when TOP1 is deleted. Finally, bacterially expressed yeast Tdp1p is able to remove a peptide derived from yTop2 that is covalently bound to DNA by a 5' phosphotyrosyl linkage. Our results show that Tdp1 plays more general roles in DNA repair than repair of Top1 mediated DNA damage, and may participate in repairing many types of base damage to DNA.

Fig. 1.

Δtdp1 cells overexpressing TOP2 enzyme are hypersensitive to etoposide. Etoposide sensitivity of Δtdp1 and wild-type strains carrying yCP50 (vector control) or a plasmid overexpressing (OE) wild-type yeast TOP2 enzyme was determined as described in Materials and Methods. The asterisk indicates a significant difference between wild type and Δtdp1 at the indicated drug concentration.

Fig. 2.

Combination of Δtdp1 with either Δku80 or Δrad6 increases sensitivity to etoposide. Etoposide sensitivity of wild-type, Δtdp1, Δku80, and Δtdp1Δku80 mutant cells overexpressing TOP2 (A) or in the absence of TOP2 overexpression (B) was determined. (C) Survival of Δtdp1, Δrad6, or Δtdp1Δrad6 strains after exposure to etoposide. An asterisk indicates a significant difference between the single mutant and the double mutant at the indicated drug concentration.

Fig. 3.

Combining mutations conferring defects in homologous recombination with Δtdp1 results in minor increases in etoposide sensitivity. Strains with deletions in Δrad52 or Δrad52Δtdp1 were examined for sensitivity to etoposide. Differences in survival between the two strains at each etoposide concentration were not significant.

Fig. 4.

Increased sensitivity of Δtdp1 to etoposide does not require TOP1. (A) Sensitivity of wild-type, Δtdp1, Δtop1, or Δtdp1Δtop1 strains to etoposide was assessed by using the same approach as in Fig. 1. An asterisk indicates a significant difference between Δtop1 and Δtop1 Δtdp1 at the indicated drug concentration. (B) Sensitivity of wild-type, Δtdp1, Δtop1 or Δtdp1Δtop1 strains to etoposide was also determined by spotting diluted cell cultures onto etoposide containing YPDA agar plates. Relevant genotypes are indicated.

Fig. 5.

Combination of Δtdp1 with deletions of nuclease genes relevant to DNA repair leads to additive sensitivity to etoposide. (A) Survival of WT, Δtdp1, Δrad1, Δrad2, Δrad1Δtdp1, and Δrad2Δtdp1 strains was determined by spotting diluted cell cultures onto etoposide containing YPDA agar plates. (B) Survival of WT, Δtdp1, Δmms4, and Δmms4Δtdp1 strains to etoposide was assessed by spotting diluted cell cultures onto etoposide containing YPDA agar plates. (C) Survival of the same strains as shown in B was assessed after 24-h etoposide exposure in liquid culture assay followed by plating to determine survival. An asterisk indicates a significant difference between Δmms4 and Δmms4Δtdp1 at the indicated drug concentration.

Fig. 6.

Removal of 5′ linked peptide bound to DNA by yeast TDP1 protein. (A) Schematic representation of Tdp1 enzymatic processing of a 5′-linked oligopeptide substrate. Top2 is covalently trapped on a 3′ end-labeled oligonucleotide duplex suicide substrate (17D) via the active site tyrosine. Digestion with trypsin leaves an 8-aa peptide bound to the labeled substrate (14D-top2-pep). If Tdp1 cleaves the peptide from DNA, the resulting product is a 14-mer oligonucleotide (14D). The peptide linked substrate used in B and C was boiled before use, and is therefore single stranded. (B) Serial 5-fold dilutions of yeast Tdp1p, starting with 1 μg protein were incubated with 0.5 fmol of substrate for 15 min (lanes 3–8) as well as in the presence of the inhibitor sodium orthovanadate (lanes 9–13). 17D is the original 3′ end-labeled suicide substrate (lane 1), and mock-treated substrate is in lane 2 (14D-top2-pep). (C) The 5′-linked oligopeptide (0.5 fmol of 14D-top2-pep) was incubated with the indicated amounts of wild-type or H182A (active site mutant) Tdp1p. The cleavage product (14D) was only observed after incubation with wild-type Tdp1p (lane 3).