Hypersensitivity of Nonhomologous DNA End-joining Mutants to VP-16 and ICRF-193

135

113

Tyrosyl DNA phosphodiesterase 1 (TDP1) is a repair enzyme that removes adducts, e.g. of topoisomerase I from the 3-phosphate of DNA breaks. When expressed in human cells as biofluorescent chimera, TDP1 appeared more mobile than topoisomerase I, less accumulated in nucleoli, and not chromosome-bound at early mitosis. Upon exposure to camptothecin both proteins were cleared from nucleoli and rendered less mobile in the nucleoplasm. However, with TDP1 this happened much more slowly reflecting most likely the redistribution of nucleolar structures upon inhibition of rDNA transcription. Thus, a steady association of TDP1 with topoisomerase I seems unlikely, whereas its integration into repair complexes assembled subsequently to the stabilization of DNA⅐topoisomerase I intermediates is supported. Cells expressing GFP-tagged TDP1 > 100-fold in excess of endogenous TDP1 exhibited a significant reduction of DNA damage induced by the topoisomerase I poison camptothecin and could be selected by that drug. Surprisingly, DNA damage induced by the topoisomerase II poison VP-16 was also diminished to a similar extent, whereas DNA damage independent of topoisomerase I or II was not affected. Overexpression of the inactive mutant GFP-TDP1 H263A at similar levels did not reduce DNA damage by camptothecin or VP-16. These observations confirm a requirement of active TDP1 for the repair of topoisomerase I-mediated DNA damage. Our data also suggest a role of TDP1 in the repair of DNA damage mediated by topoisomerase II, which is less clear. Since overexpression of TDP1 did not compromise cell proliferation, it could be a pleiotropic resistance mechanism in cancer therapy.Tyrosyl DNA phosphodiesterase 1 (TDP1) 1 is an enzyme capable of hydrolyzing phosphodiester bonds between tyrosine and the 3Ј-phosphate of DNA (1, 2), which are typically generated in a transient manner by DNA topoisomerase I (topo I) (3). In keeping with this, yeast deletion mutations of TDP1 are deficient in the repair of DNA damage induced by camptothecin, a drug that stabilizes the transient topo I⅐DNA intermediate (2, 4 -6). More precisely, TDP1 has been characterized in these studies as a non-exclusive effector upstream of Rad52 that removes structurally modified topo I adducts (7, 8) as well as oxidative adducts (9) from the 3Ј-phosphate of a DNA break prior to homologous recombination repair. In mammals, TDP1 is (in addition or instead?) involved in an XRCC-dependent single-stranded DNA repair pathway also directed at topo I⅐DNA adducts (10 -12). Despite all the evidence of yeast deletion studies implying TDP1 in DNA repair, a familial disease caused by a mutation in the active site of the human ortholog of the enzyme exhibits a phenotype not at all typical for inadequate DNA repair, namely a slow onset of neuronal degeneration (13). This unexpected finding has prompted speculations that at least in mammals TDP1 could serve a much broader scope of functions, some of which may not even depend on catalytic activity. To further clarify the importance of TDP1 for...

Section: Discussionmentioning

confidence: 99%

TDP1 Overexpression in Human Cells Counteracts DNA Damage Mediated by Topoisomerases I and II

Barthelmes

Habermeyer

Christensen

et al. 2004

135

113

“…The idea that efficient alternative pathways (other than HR) may work to repair Top1-mediated DNA damage contrasts with the situation in yeast where HR plays essential roles in Top1 damage repair (28); specifically, several redundant pathways are involved in the initial processing of Top1 damage, but all the pathways eventually rely on HR to complete repair. Yet, even the partial contribution from HR in vertebrate Top1 damage repair would be intriguing in light of our recent finding that vertebrate Top2-mediated DNA damage (induced by Top2 inhibitors) is predominantly repaired by NHEJ, not HR (21). In yeast, HR is responsible for the repair of Top2-mediated DNA damage, whereas NHEJ has only a minor role (49).…”

Section: Discussionmentioning

confidence: 99%

“…Inhibition of the enzyme by Top2 inhibitors, such as VP-16 or ICRF-193, has been shown to generate DSBs (17)(18)(19)(20). We have recently shown that Top2-mediated DNA damage is predominantly repaired by the NHEJ pathway as evidenced by the observations that NHEJ mutants (LIG4 Ϫ/Ϫ and KU70 Ϫ/Ϫ cells) are extremely sensitive to Top2 inhibitors compared with wild-type or RAD54 Ϫ/Ϫ cells (21). Interestingly, the absence of Rad54 alleviated the hypersensitivity of KU70 Ϫ/Ϫ cells, suggesting that the cytotoxicity of Top2 inhibitors is enhanced by HR (21).…”

mentioning

confidence: 99%

“…We have recently shown that Top2-mediated DNA damage is predominantly repaired by the NHEJ pathway as evidenced by the observations that NHEJ mutants (LIG4 Ϫ/Ϫ and KU70 Ϫ/Ϫ cells) are extremely sensitive to Top2 inhibitors compared with wild-type or RAD54 Ϫ/Ϫ cells (21). Interestingly, the absence of Rad54 alleviated the hypersensitivity of KU70 Ϫ/Ϫ cells, suggesting that the cytotoxicity of Top2 inhibitors is enhanced by HR (21). On the other hand, Helleday and co-workers (22,23) have reported that HR plays a more prominent role in the repair of replication fork-associated DNA damage than does NHEJ.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Loss of Nonhomologous End Joining Confers Camptothecin Resistance in DT40 Cells

Adachi

Koyama

2004

Self Cite

DNA topoisomerase I (Top1) generates transient DNA single-strand breaks via the formation of cleavage complexes in which the enzyme is linked to the 3 -phosphate of the cleavage strand. The anticancer drug camptothecin (CPT) poisons Top1 by trapping cleavage complexes, thereby inducing Top1-linked single-strand breaks. Such DNA lesions are converted into DNA double-strand breaks (DSBs) upon collision with replication forks, implying that DSB repair pathways could be involved in the processing/repair of Top1-mediated DNA damage. Here we report that Top1-mediated DNA damage is repaired primarily by homologous recombination, a major pathway of DSB repair. Unexpectedly, however, we found that nonhomologous end joining (NHEJ), another DSB repair pathway, has no positive role in the relevant repair; notably, DT40 cell mutants lacking either of the NHEJ factors (namely, Ku70, DNA-dependent protein kinase catalytic subunit, and DNA ligase IV) were resistant to killing by CPT. In addition, we showed that the absence of NHEJ alleviates the requirement of homologous recombination in the repair of CPT-induced DNA damage. Our results indicate that NHEJ can be a cytotoxic pathway in the presence of CPT, shedding new light on the molecular mechanisms for the formation and repair of Top1-mediated DNA damage in vertebrates. Thus, our data have significant implications for cancer chemotherapy involving Top1 inhibitors.DNA double-strand breaks (DSBs) 1 can be caused by a variety of exogenous and endogenous agents, posing a major threat to genome integrity. If left unrepaired, DSBs may cause cell death (1, 2). Vertebrate cells have evolved two distinct pathways for repairing DSBs, homologous recombination (HR) and nonhomologous end joining (NHEJ) (2-5). The HR reaction requires a wide variety of proteins including Rad51, Rad52, and Rad54 (2), whereas NHEJ relies on Ku (a heterodimer of Ku70 and Ku86), DNA-PKcs, Artemis, Xrcc4, and DNA ligase IV (the LIG4 gene product) (3, 5, 6). The requirement for DNA ligase IV in this pathway is exclusive as other DNA ligases (I and III) are unable to substitute for the ligase function (7,8). In contrast to HR that allows for accurate repair of DSBs, NHEJ is typically an imprecise, error-prone pathway.In accordance with the essential roles of HR and NHEJ in DSB repair, cells deficient in HR or NHEJ have been shown to be highly sensitive to ionizing radiation (7-14). In the chicken B-lymphocyte DT40 cell line (15), the extent of radiosensitivity of RAD54 Ϫ/Ϫ cells is similar to that of DNA-PKcs Ϫ/Ϫ/Ϫ or LIG4 Ϫ/Ϫ cells (8, 14), and RAD54 Ϫ/Ϫ /DNA-PKcs Ϫ/Ϫ/Ϫ double mutant cells are much more radiosensitive than each single mutant (14). These results clearly indicate that HR and NHEJ contribute equally, and independently, to the repair of radiation-induced DSBs.Perturbation of fundamental cellular processes such as DNA replication or the action of DNA topoisomerases often causes DSBs. DNA topoisomerases are ubiquitous nuclear enzymes that participate in many aspects of DNA metabolisms, including DNA ...

“…In this respect, we previously reported that Top2-mediated DNA damage is predominantly repaired by the non-homologous end-joining (NHEJ) pathway in higher eukaryotes, contrasting with the situation in yeast, where homologous recombination repairs virtually all Top2-mediated DNA damage (37,39,46,47). In mammalian cells, NHEJ and homologous recombination are the two major pathways of DSB repair, and these pathways are roughly equally important for the repair of radiation-induced DSBs (48 -51).…”

Section: Nk314mentioning

confidence: 99%

NK314, a Topoisomerase II Inhibitor That Specifically Targets the α Isoform

Toyoda

Kagaya

Cowell

et al. 2008

Self Cite

108

Topoisomerase II (Top2) is a ubiquitous nuclear enzyme that relieves torsional stress in chromosomal DNA during various cellular processes. Agents that target Top2, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective anticancer drugs used in the clinic. Mammalian cells possess two genetically distinct Top2 isoforms, both of which are the target of these agents. Top2␣ is essential for cell proliferation and is highly expressed in vigorously growing cells, whereas Top2␤ is nonessential for growth and has recently been implicated in treatment-associated secondary malignancies, highlighting the validity of a Top2␣-specific drug for future cancer treatment; however, no such agent has been hitherto reported. Here we show that NK314, a novel synthetic benzo[c]phenanthridine alkaloid, targets Top2␣ and not Top2␤ in vivo. Unlike other Top2 inhibitors, NK314 induces Top2-DNA complexes and double-strand breaks (DSBs) in an ␣ isoform-specific manner. Heterozygous disruption of the human TOP2␣ gene confers increased NK314 resistance, whereas TOP2␤ homozygous knock-out cells display increased NK314 sensitivity, indicating that the ␣ isoform is the cellular target. We further show that the absence of Top2␤ does not alleviate NK314 hypersensitivity of cells deficient in non-homologous end-joining, a critical pathway for repairing Top2-mediated DSBs. Our results indicate that NK314 acts as a Top2␣-specific poison in mammalian cells, with excellent potential as an efficacious and safe chemotherapeutic agent. We also suggest that a series of human knock-out cell lines are useful in assessing DNA damage and repair induced by potential topoisomerase-targeting agents. DNA topoisomerase II (Top2)2 is a ubiquitous nuclear enzyme that alters the topological structure of DNA and chromosomes through a transient DNA double-strand break (DSB) and subsequent religation of the DSB (1, 2). The enzyme has been implicated in many aspects of DNA metabolism, including DNA replication, repair, transcription, and chromosome condensation/segregation (1, 3). Top2 has been of considerable interest to human medicine, because it is an important target for cancer chemotherapy (4). Top2-targeting agents, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective and widely used anticancer drugs in cancer chemotherapy (5, 6). These agents are referred to as "Top2 poisons," because they convert the essential enzyme into a highly cytotoxic DNA-damaging agent through the formation of "cleavage complex" (also called "cleavable complex"), in which a Top2-linked DNA strand-passing intermediate is stabilized, allowing the generation of a DSB (7,8).Mammalian cells possess two genetically distinct Top2 isoforms (9, 10). Despite their similar structural features (ϳ70% identity at the amino acid level) and biological properties, the two isoforms are differentially regulated and play different roles in living cells. Top2␣ is most abundantly expressed in rapidly growing tissues and its expression is cell cycle-regulated...