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.D NA topoisomerases (topos) carry out alterations in DNA structure by transiently introducing breaks in DNA. Topos use tyrosine residues to form a transient covalent intermediate between the enzyme and DNA, resulting in single or double strand DNA breaks (1). This mechanism of DNA breakage can create difficulties for a cell under conditions that interfere with the normal religation reaction of the enzyme, such as the presence of specific inhibitors. Then, the enzyme becomes trapped on DNA as a stable covalent adduct (2). Topos can become trapped by small molecule inhibitors such as camptothecin or etoposide, which are clinically used anticancer agents targeting topo I and topo II, respectively. The complete repair of this type of lesion requires DNA strand break repair pathways, and also requires activities that can remove covalent protein DNA adducts (3).An enzyme described by Nash and colleagues (4), tyrosyl-DNA phosphodiesterase (Tdp1), can remove peptides covalently bound to DNA by a 3Ј phosphotyrosine linkage. This enzyme participates in the repair of Top1 covalently trapped on DNA in the presence of camptothecin in both yeast (5, 6) and mammalian cells (7). Tdp1 is a phospholipase D superfamily enzyme (8), and the three-dimensional structure of the protein (9) as well as transition state intermediates have been reported (10). Tdp1 does not define the only pathway for removing Top1 that is trapped by camptothecin, ...

Spermiogenesis and DNA Repair: A Possible Etiology of Human Infertility and Genetic Disorders

“…Using confocal microscopy, we recently confirmed the presence of topoisomerase IIb (TOP2B, topoIIb) distributed in foci during steps 9 to 13 of mouse spermiogenesis but did not observe the presence of the a isoform at these steps [Leduc et al unpublished results]. Interestingly, we identified the presence of tyrosyl-DNA phosphodiesterase 1(TDP1), an enzyme known to resolve topoisomerasesmediated DNA damage [Interthal et al 2005;Nitiss et al 2006;Raymond and Burgin 2006]. The presence of TDP1 is coincident with the appearance of topoIIb in ES.…”

Tyrosyl-DNA Phosphodiesterase as a Target for Anticancer Therapy

“…Yeast Tdp1 has also been shown to process a topoisomerase II (Top2) cleavage complex or a 5′-phosphotyrosyl bond [53]. Yet, the effects of Top2 inhibitors on TDP1-deficient yeast cells have been contradictory [53,54]. Moreover, a single study in mammalian cells has demonstrated that overexpression of an active Tdp1 significantly reduces the DNA damage caused by Top2 inhibitors [26].…”

“…However, it is interesting to note that the Tdp1 is only able to remove a single terminal 3′-mononucleoside and is incapable of processing a DNA end containing a 3′-phosphate [52], which demonstrates that Tdp1 cannot act as a 3′-exonuclease. Yeast Tdp1 has also been shown to process a topoisomerase II (Top2) cleavage complex or a 5′-phosphotyrosyl bond [53]. Yet, the effects of Top2 inhibitors on TDP1-deficient yeast cells have been contradictory [53,54].…”

Role of tyrosyl-DNA phosphodiesterase (TDP1) in mitochondria

“…Thus, TDP1 may function to remove a variety of adducts from 3′-DNA ends during DNA repair (50). Yeast TDP1 has also been shown to process Top2-DNA adducts (51).…”