Optimal function of the DNA repair enzyme TDP1 requires its phosphorylation by ATM and/or DNA-PK.
ABSTRACT: Human tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond at a DNA 3' end linked to a tyrosyl moiety. This type of linkage is found at stalled topoisomerase I (Top1)-DNA covalent complexes, and TDP1 has been implicated in the repair of such complexes. Here we show that Top1-associated DNA double-stranded breaks (DSBs) induce the phosphorylation of TDP1 at S81. This phosphorylation is mediated by the protein kinases: ataxia-telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK). Phosphorylated TDP1 forms nuclear foci that co-localize with those of phosphorylated histone H2AX (gammaH2AX). Both Top1-induced replication- and transcription-mediated DNA damages induce TDP1 phosphorylation. Furthermore, we show that S81 phosphorylation stabilizes TDP1, induces the formation of XRCC1 (X-ray cross-complementing group 1)-TDP1 complexes and enhances the mobilization of TDP1 to DNA damage sites. Finally, we provide evidence that TDP1-S81 phosphorylation promotes cell survival and DNA repair in response to CPT-induced DSBs. Together; our findings provide a new mechanism for TDP1 post-translational regulation by ATM and DNA-PK.
Project description:Although defective repair of DNA double-strand breaks (DSBs) leads to neurodegenerative diseases, the processes underlying their production and signaling in non-replicating cells are largely unknown. Stabilized topoisomerase I cleavage complexes (Top1cc) by natural compounds or common DNA alterations are transcription-blocking lesions whose repair depends primarily on Top1 proteolysis and excision by tyrosyl-DNA phosphodiesterase-1 (TDP1). We previously reported that stabilized Top1cc produce transcription-dependent DSBs that activate ATM in neurons. Here, we use camptothecin (CPT)-treated serum-starved quiescent cells to induce transcription-blocking Top1cc and show that those DSBs are generated during Top1cc repair from Top1 peptide-linked DNA single-strand breaks generated after Top1 proteolysis and before excision by TDP1. Following DSB induction, ATM activates DNA-PK whose inhibition suppresses H2AX and H2A ubiquitination and the later assembly of activated ATM into nuclear foci. Inhibition of DNA-PK also reduces Top1 ubiquitination and proteolysis as well as resumption of RNA synthesis suggesting that DSB signaling further enhances Top1cc repair. Finally, we show that co-transcriptional DSBs kill quiescent cells. Together, these new findings reveal that DSB production and signaling by transcription-blocking Top1 lesions impact on non-replicating cell fate and provide insights on the molecular pathogenesis of neurodegenerative diseases such as SCAN1 and AT syndromes, which are caused by TDP1 and ATM deficiency, respectively.
Project description:Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3'-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1-PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1.
Project description:Tyrosyl-DNA phosphodiesterase I (TDP1) repairs stalled topoisomerase I (Top1)-DNA covalent complexes and has been proposed to be a promising and attractive target for cancer treatment. Inhibitors of TDP1 could conceivably act synergistically with Top1 inhibitors and thereby potentiate the effects of Top1 poisons. This study describes the successful design and synthesis of 2-position-modified indenoisoquinolines as dual Top1-TDP1 inhibitors using a structure-based drug design approach. Enzyme inhibition studies indicate that indenoisoquinolines modified at the 2-position with three-carbon side chains ending with amino substituents show both promising Top1 and TDP1 inhibitory activity. Molecular modeling of selected target compounds bound to Top1 and TDP1 was used to rationalize the enzyme inhibition results and structure-activity relationship analysis.
Project description:Tyrosyl-DNA-phosphodiesterase 1 (TDP1) repairs 3'-blocking DNA lesions by catalytically hydrolyzing the tyrosyl-DNA-phosphodiester bond of trapped topoisomerase I (Top1) cleavage complexes (Top1cc). It also removes 3'-blocking residues derived from oxidative damage or incorporation of chain terminating anticancer and antiviral nucleosides. Thus, TDP1 is regarded as a determinant of resistance to Top1 inhibitors and chain terminating nucleosides, and possibly of genomic stability. In the 60 cell lines of the NCI Developmental Therapeutic Anticancer Screen (the NCI-60), whose whole genome transcriptome and mutations have recently been characterized, we discovered two human lung cancer cell lines deficient for TDP1 (NCI_H522 and HOP_62). HOP_62 shows undetectable TDP1 mRNA and NCI_H522 bears a homozygous deleterious mutation of TDP1 at a highly conserved amino acid residue (K292E). Absence of TDP1 protein and lack of TDP1 catalytic activity were demonstrated in cell lysates from both cell lines. Lack of TDP1 expression in HOP_62 was shown to be due to TDP1 promoter hypermethylation. Our study provides insights into the possible inactivation of TDP1 in cancers and its relationship to cellular response to Top1-targeted drugs. It also reveals two TDP1 knockout lung cancer cell lines for further TDP1 functional analyses.
Project description:A homozygous mutation of human tyrosyl-DNA phosphodiesterase 1 (TDP1) causes the neurodegenerative syndrome, spinocerebellar ataxia with axonal neuropathy (SCAN1). TDP1 hydrolyzes the phosphodiester bond between DNA 3'-end and a tyrosyl moiety within trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). TDP1 is critical for mitochondrial DNA (mtDNA) repair; however, the role of mitochondria remains largely unknown for the etiology of SCAN1. We demonstrate that mitochondria in cells expressing SCAN1-TDP1 (TDP1H493R) are selectively trapped on mtDNA in the regulatory non-coding region and promoter sequences. Trapped TDP1H493R-mtDNA complexes were markedly increased in the presence of the Top1 poison (mito-SN38) when targeted selectively into mitochondria in nanoparticles. TDP1H493R-trapping accumulates mtDNA damage and triggers Drp1-mediated mitochondrial fission, which blocks mitobiogenesis. TDP1H493R prompts PTEN-induced kinase 1-dependent mitophagy to eliminate dysfunctional mitochondria. SCAN1-TDP1 in mitochondria creates a pathological state that allows neurons to turn on mitophagy to rescue fit mitochondria as a mechanism of survival.
Project description:Inhibitors of topoisomerase I (Top1) that result in stalled Top1 cleavage complexes (Top1cc) are commonly employed against cancer. Combination chemotherapy with DNA repair inhibitors can potentially improve response to these widely used chemotherapeutics. One line of inquiry focuses on inhibitors of tyrosyl-DNA phosphodiesterase 1 (Tdp1), a repair enzyme for Top1cc. Tdp1 catalyzes the hydrolysis of DNA adducts covalently linked to the 3'-phosphate of DNA, including Top1-derived peptides and also 3'-phosphoglycolates. Tdp1 inhibitors should synergize not only with Top1-targeting drugs (camptothecins, indenoisoquinolines), but also with bleomycin, topoisomerase II (Top2) inhibitors (etoposide, doxorubicin) and DNA alkylating agents. Here, we summarize the structure-activity relationship obtained from the reported Tdp1 inhibitors. Better understanding of Top1cc repair in vivo coupled with detailed structural studies on Tdp1-inhibitor interaction will be crucial in guiding the rational design of Tdp1 inhibitors.
Project description:Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a recently discovered enzyme that catalyzes the hydrolysis of 3'-phosphotyrosyl bonds. Such linkages form in vivo following the DNA processing activity of topoisomerase I (Top1). For this reason, Tdp1 has been implicated in the repair of irreversible Top1-DNA covalent complexes, which can be generated by either exogenous or endogenous factors. Tdp1 has been regarded as a potential therapeutic co-target of Top1 in that it seemingly counteracts the effects of Top1 inhibitors, such as camptothecin and its clinically used derivatives. Thus, by reducing the repair of Top1-DNA lesions, Tdp1 inhibitors have the potential to augment the anticancer activity of Top1 inhibitors provided there is a presence of genetic abnormalities related to DNA checkpoint and repair pathways. Human Tdp1 can also hydrolyze other 3'-end DNA alterations including 3'-phosphoglycolates and 3'-abasic sites indicating it may function as a general 3'-DNA phosphodiesterase and repair enzyme. The importance of Tdp1 in humans is highlighted by the observation that a recessive mutation in the human TDP1 gene is responsible for the inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1). This review provides a summary of the biochemical and cellular processes performed by Tdp1 as well as the rationale behind the development of Tdp1 inhibitors for anticancer therapy.
Project description:Tyrosyl-DNA phosphodiesterase I (Tdp1) is a cellular enzyme that repairs the irreversible topoisomerase I (Top1)-DNA complexes and confers chemotherapeutic resistance to Top1 inhibitors. Inhibiting Tdp1 provides an attractive approach to potentiating clinically used Top1 inhibitors. However, despite recent efforts in studying Tdp1 as a therapeutic target, its inhibition remains poorly understood and largely underexplored. We describe herein the discovery of arylidene thioxothiazolidinone as a scaffold for potent Tdp1 inhibitors based on an initial tyrphostin lead compound 8. Through structure-activity relationship (SAR) studies we demonstrated that arylidene thioxothiazolidinones inhibit Tdp1 and identified compound 50 as a submicromolar inhibitor of Tdp1 (IC?? = 0.87 ?M). Molecular modeling provided insight into key interactions essential for observed activities. Some derivatives were also active against endogenous Tdp1 in whole cell extracts. These findings contribute to advancing the understanding on Tdp1 inhibition.
Project description:Human tyrosyl-DNA phosphodiesterases (TDP) hydrolyze the phosphodiester bond between DNA and the catalytic tyrosine of Top1 to excise topoisomerase I cleavage complexes (Top1cc) that are trapped by camptothecin (CPT) and by genotoxic DNA alterations. Here we show that the protein arginine methyltransferase PRMT5 enhances the repair of Top1cc by direct binding to TDP1 and arginine dimethylation of TDP1 at residues R361 and R586. Top1-induced replication-mediated DNA damage induces TDP1 arginine methylation, enhancing its 3'- phosphodiesterase activity. TDP1 arginine methylation also increases XRCC1 association with TDP1 in response to CPT, and the recruitment of XRCC1 to Top1cc DNA damage foci. PRMT5 knockdown cells exhibit defective TDP1 activity with marked elevation in replication-coupled CPT-induced DNA damage and lethality. Finally, methylation of R361 and R586 stimulate TDP1 repair function and promote cell survival in response to CPT. Together, our findings provide evidence for the importance of PRMT5 for the post-translational regulation of TDP1 and repair of Top1cc.
Project description:The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3'- or 5'-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase 'poisons'. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3'-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3'-tyrosyl DNA phosphodiesterase (3'-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1-/- cells, suggesting that Tdp2 contributes to cellular 3'-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1-/-/Tdp2-/-/- DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1-/- DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a contribution for Tdp1 to repair Top2-mediated damage. Together, our data suggest that Tdp1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.