Publications by authors named "Shar-Yin N Huang"

23 Publications

  • Page 1 of 1

SLFN11 inactivation induces proteotoxic stress and sensitizes cancer cells to ubiquitin activating enzyme inhibitor TAK-243.

Cancer Res 2021 Apr 16. Epub 2021 Apr 16.

Center for Cancer Research, Developmental Therapeutics Branch & Laboratory of Molecular Pharmacology, National Cancer Institute

Schlafen11 (SLFN11) inactivation occurs in approximately 50% of cancer cell lines and in a large fraction of patient tumor samples, which leads to chemoresistance. Therefore, new therapeutic approaches are needed to target SLFN11-deficient cancers. To that effect, we conducted a drug screen with the NCATS mechanistic drug library of 1978 compounds in isogenic SLFN11-knockout (KO) and wild-type (WT) leukemia cell lines. Here we report that TAK-243, a first-in-class ubiquitin activating enzyme UBA1 inhibitor in clinical development, causes preferential cytotoxicity in SLFN11-KO cells; this effect is associated with claspin-mediated DNA replication inhibition by CHK1 independently of ATR. Additional analyses showed that SLFN11-KO cells exhibit consistently enhanced global protein ubiquitylation, endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and protein aggregation. TAK-243 suppressed global protein ubiquitylation and activated the UPR transducers PERK, phosphorylated eIF2alpha, phosphorylated IRE1, and ATF6 more effectively in SLFN11-KO cells than WT cells. Proteomic analysis using biotinylated mass spectrometry and RNAi screening also showed physical and functional interactions of SLFN11 with translation initiation complexes and protein folding machinery. These findings uncover a previously unknown function of SLFN11 as a regulator of protein quality control and attenuator of ER stress and UPR. Moreover, they suggest the potential value of TAK-243 in SLFN11-deficient tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-20-2694DOI Listing
April 2021

Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes.

Sci Adv 2021 Mar 3;7(10). Epub 2021 Mar 3.

Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.

The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5'-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.abe0384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929499PMC
March 2021

The Indenoisoquinoline LMP517: A Novel Antitumor Agent Targeting both TOP1 and TOP2.

Mol Cancer Ther 2020 08 19;19(8):1589-1597. Epub 2020 May 19.

Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland.

The camptothecin derivatives topoisomerase I (TOP1) inhibitors, irinotecan and topotecan, are FDA approved for the treatment of colorectal, ovarian, lung and breast cancers. Because of the chemical instability of camptothecins, short plasma half-life, drug efflux by the multidrug-resistance ABC transporters, and the severe diarrhea produced by irinotecan, indenoisoquinoline TOP1 inhibitors (LMP400, LMP776, and LMP744), which overcome these limitations, have been developed and are in clinical development. Further modifications of the indenoisoquinolines led to the fluoroindenoisoquinolines, one of which, LMP517, is the focus of this study. LMP517 showed better antitumor activity than its parent compound LMP744 against H82 (small cell lung cancer) xenografts. Genetic analyses in DT40 cells showed a dual TOP1 and TOP2 signature with selectivity of LMP517 for DNA repair-deficient tyrosyl DNA phosphodiesterase 2 (TDP2)- and Ku70-knockout cells. RADAR assays revealed that LMP517, and to a lesser extent LMP744, induce TOP2 cleavage complexes (TOP2cc) in addition to TOP1ccs. Histone γH2AX detection showed that, unlike classical TOP1 inhibitors, LMP517 targets cells independently of their position in the cell cycle. Our study establishes LMP517 as a dual TOP1 and TOP2 inhibitor with therapeutic potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-19-1064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415565PMC
August 2020

Topoisomerase II-Induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity.

Mol Cell 2019 07 12;75(2):252-266.e8. Epub 2019 Jun 12.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA. Electronic address:

Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2019.04.030DOI Listing
July 2019

TDP1 suppresses mis-joining of radiomimetic DNA double-strand breaks and cooperates with Artemis to promote optimal nonhomologous end joining.

Nucleic Acids Res 2018 09;46(17):8926-8939

Department of Pharmacology and Toxicology and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.

The Artemis nuclease and tyrosyl-DNA phosphodiesterase (TDP1) are each capable of resolving protruding 3'-phosphoglycolate (PG) termini of DNA double-strand breaks (DSBs). Consequently, both a knockout of Artemis and a knockout/knockdown of TDP1 rendered cells sensitive to the radiomimetic agent neocarzinostatin (NCS), which induces 3'-PG-terminated DSBs. Unexpectedly, however, a knockdown or knockout of TDP1 in Artemis-null cells did not confer any greater sensitivity than either deficiency alone, indicating a strict epistasis between TDP1 and Artemis. Moreover, a deficiency in Artemis, but not TDP1, resulted in a fraction of unrepaired DSBs, which were assessed as 53BP1 foci. Conversely, a deficiency in TDP1, but not Artemis, resulted in a dramatic increase in dicentric chromosomes following NCS treatment. An inhibitor of DNA-dependent protein kinase, a key regulator of the classical nonhomologous end joining (C-NHEJ) pathway sensitized cells to NCS, but eliminated the sensitizing effects of both TDP1 and Artemis deficiencies. These results suggest that TDP1 and Artemis perform different functions in the repair of terminally blocked DSBs by the C-NHEJ pathway, and that whereas an Artemis deficiency prevents end joining of some DSBs, a TDP1 deficiency tends to promote DSB mis-joining.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gky694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158748PMC
September 2018

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2 short isoform.

EMBO Rep 2018 03 9;19(3). Epub 2018 Feb 9.

Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2) expressed from an alternative transcription start site. TDP2 contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type () and knockout () DT40 cells shows that cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing cells with human restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in cells. Using CRISPR-engineered human cells expressing only the TDP2 isoform, we show that TDP2 also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2 isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.201642139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836098PMC
March 2018

Topoisomerase I-mediated cleavage at unrepaired ribonucleotides generates DNA double-strand breaks.

EMBO J 2017 02 8;36(3):361-373. Epub 2016 Dec 8.

Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA

Ribonuclease activity of topoisomerase I (Top1) causes DNA nicks bearing 2',3'-cyclic phosphates at ribonucleotide sites. Here, we provide genetic and biochemical evidence that DNA double-strand breaks (DSBs) can be directly generated by Top1 at sites of genomic ribonucleotides. We show that RNase H2-deficient yeast cells displayed elevated frequency of Rad52 foci, inactivation of RNase H2 and RAD52 led to synthetic lethality, and combined loss of RNase H2 and RAD51 induced slow growth and replication stress. Importantly, these phenotypes were rescued upon additional deletion of TOP1, implicating homologous recombination for the repair of Top1-induced damage at ribonuclelotide sites. We demonstrate biochemically that irreversible DSBs are generated by subsequent Top1 cleavage on the opposite strand from the Top1-induced DNA nicks at ribonucleotide sites. Analysis of Top1-linked DNA from pull-down experiments revealed that Top1 is covalently linked to the end of DNA in RNase H2-deficient yeast cells, supporting this model. Taken together, these results define Top1 as a source of DSBs and genome instability when ribonucleotides incorporated by the replicative polymerases are not removed by RNase H2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201592426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286372PMC
February 2017

Roles of eukaryotic topoisomerases in transcription, replication and genomic stability.

Nat Rev Mol Cell Biol 2016 11 21;17(11):703-721. Epub 2016 Sep 21.

College of Pharmacy, University of Illinois, Rockford, Illinois 61107, USA.

Topoisomerases introduce transient DNA breaks to relax supercoiled DNA, remove catenanes and enable chromosome segregation. Human cells encode six topoisomerases (TOP1, TOP1mt, TOP2α, TOP2β, TOP3α and TOP3β), which act on a broad range of DNA and RNA substrates at the nuclear and mitochondrial genomes. Their catalytic intermediates, the topoisomerase cleavage complexes (TOPcc), are therapeutic targets of various anticancer drugs. TOPcc can also form on damaged DNA during replication and transcription, and engage specific repair pathways, such as those mediated by tyrosyl-DNA phosphodiesterase 1 (TDP1) and TDP2 and by endonucleases (MRE11, XPF-ERCC1 and MUS81). Here, we review the roles of topoisomerases in mediating chromatin dynamics, transcription, replication, DNA damage repair and genomic stability, and discuss how deregulation of topoisomerases can cause neurodegenerative diseases, immune disorders and cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nrm.2016.111DOI Listing
November 2016

Parallel analysis of ribonucleotide-dependent deletions produced by yeast Top1 in vitro and in vivo.

Nucleic Acids Res 2016 09 1;44(16):7714-21. Epub 2016 Jun 1.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA

Ribonucleotides are the most abundant non-canonical component of yeast genomic DNA and their persistence is associated with a distinctive mutation signature characterized by deletion of a single repeat unit from a short tandem repeat. These deletion events are dependent on DNA topoisomerase I (Top1) and are initiated by Top1 incision at the relevant ribonucleotide 3'-phosphodiester. A requirement for the re-ligation activity of Top1 led us to propose a sequential cleavage model for Top1-dependent mutagenesis at ribonucleotides. Here, we test key features of this model via parallel in vitro and in vivo analyses. We find that the distance between two Top1 cleavage sites determines the deletion size and that this distance is inversely related to the deletion frequency. Following the creation of a gap by two Top1 cleavage events, the tandem repeat provides complementarity that promotes realignment to a nick and subsequent Top1-mediated ligation. Complementarity downstream of the gap promotes deletion formation more effectively than does complementarity upstream of the gap, consistent with constraints to realignment of the strand to which Top1 is covalently bound. Our data fortify sequential Top1 cleavage as the mechanism for ribonucleotide-dependent deletions and provide new insight into the component steps of this process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkw495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027487PMC
September 2016

Lack of mitochondrial topoisomerase I (TOP1mt) impairs liver regeneration.

Proc Natl Acad Sci U S A 2015 Sep 24;112(36):11282-7. Epub 2015 Aug 24.

Developmental Therapeutics Branch, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892;

The liver has an exceptional replicative capacity following partial hepatectomy or chemical injuries. Cellular proliferation requires increased production of energy and essential metabolites, which critically depend on the mitochondria. To determine whether Top1mt, the vertebrate mitochondrial topoisomerase, is involved in this process, we studied liver regeneration after carbon tetrachloride (CCl4) administration. TOP1mt knockout (KO) mice showed a marked reduction in regeneration and hepatocyte proliferation. The hepatic mitochondrial DNA (mtDNA) failed to increase during recovery from CCl4 exposure. Reduced glutathione was also depleted, indicating increased reactive oxygen species (ROS). Steady-state levels of ATP, O2 consumption, mtDNA, and mitochondrial mass were also reduced in primary hepatocytes from CCl4-treated KO mice. To further test whether Top1mt acted by enabling mtDNA regeneration, we tested TOP1mt KO fibroblasts and human colon carcinoma HCT116 cells and measured mtDNA after 3-d treatment with ethidium bromide. Both types of TOP1mt knockout cells showed defective mtDNA regeneration following mtDNA depletion. Our study demonstrates that Top1mt is required for normal mtDNA homeostasis and for linking mtDNA expansion with hepatocyte proliferation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1511016112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568654PMC
September 2015

Biochemical assays for the discovery of TDP1 inhibitors.

Mol Cancer Ther 2014 Aug 14;13(8):2116-26. Epub 2014 Jul 14.

Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute;

Drug screening against novel targets is warranted to generate biochemical probes and new therapeutic drug leads. TDP1 and TDP2 are two DNA repair enzymes that have yet to be successfully targeted. TDP1 repairs topoisomerase I-, alkylation-, and chain terminator-induced DNA damage, whereas TDP2 repairs topoisomerase II-induced DNA damage. Here, we report the quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository using recombinant human TDP1. We also developed a secondary screening method using a multiple loading gel-based assay where recombinant TDP1 is replaced by whole cell extract (WCE) from genetically engineered DT40 cells. While developing this assay, we determined the importance of buffer conditions for testing TDP1, and most notably the possible interference of phosphate-based buffers. The high specificity of endogenous TDP1 in WCE allowed the evaluation of a large number of hits with up to 600 samples analyzed per gel via multiple loadings. The increased stringency of the WCE assay eliminated a large fraction of the initial hits collected from the qHTS. Finally, inclusion of a TDP2 counter-screening assay allowed the identification of two novel series of selective TDP1 inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-13-0952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4126877PMC
August 2014

Tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2).

DNA Repair (Amst) 2014 Jul 22;19:114-29. Epub 2014 May 22.

Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Building 37, Room 5068, NIH, Bethesda, MD 20892, USA.

TDP1 and TDP2 were discovered and named based on the fact they process 3'- and 5'-DNA ends by excising irreversible protein tyrosyl-DNA complexes involving topoisomerases I and II, respectively. Yet, both enzymes have an extended spectrum of activities. TDP1 not only excises trapped topoisomerases I (Top1 in the nucleus and Top1mt in mitochondria), but also repairs oxidative damage-induced 3'-phosphoglycolates and alkylation damage-induced DNA breaks, and excises chain terminating anticancer and antiviral nucleosides in the nucleus and mitochondria. The repair function of TDP2 is devoted to the excision of topoisomerase II- and potentially topoisomerases III-DNA adducts. TDP2 is also essential for the life cycle of picornaviruses (important human and bovine pathogens) as it unlinks VPg proteins from the 5'-end of the viral RNA genome. Moreover, TDP2 has been involved in signal transduction (under the former names of TTRAP or EAPII). The DNA repair partners of TDP1 include PARP1, XRCC1, ligase III and PNKP from the base excision repair (BER) pathway. By contrast, TDP2 repair functions are coordinated with Ku and ligase IV in the non-homologous end joining pathway (NHEJ). This article summarizes and compares the biochemistry, functions, and post-translational regulation of TDP1 and TDP2, as well as the relevance of TDP1 and TDP2 as determinants of response to anticancer agents. We discuss the rationale for developing TDP inhibitors for combinations with topoisomerase inhibitors (topotecan, irinotecan, doxorubicin, etoposide, mitoxantrone) and DNA damaging agents (temozolomide, bleomycin, cytarabine, and ionizing radiation), and as novel antiviral agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2014.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4090310PMC
July 2014

Proteolytic degradation of topoisomerase II (Top2) enables the processing of Top2·DNA and Top2·RNA covalent complexes by tyrosyl-DNA-phosphodiesterase 2 (TDP2).

J Biol Chem 2014 Jun 7;289(26):17960-9. Epub 2014 May 7.

From the Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892,

Eukaryotic type II topoisomerases (Top2α and Top2β) are homodimeric enzymes; they are essential for altering DNA topology by the formation of normally transient double strand DNA cleavage. Anticancer drugs (etoposide, doxorubicin, and mitoxantrone) and also Top2 oxidation and DNA helical alterations cause potentially irreversible Top2·DNA cleavage complexes (Top2cc), leading to Top2-linked DNA breaks. Top2cc are the therapeutic mechanism for killing cancer cells. Yet Top2cc can also generate recombination, translocations, and apoptosis in normal cells. The Top2 protein-DNA covalent complexes are excised (in part) by tyrosyl-DNA-phosphodiesterase 2 (TDP2/TTRAP/EAP2/VPg unlinkase). In this study, we show that irreversible Top2cc induced in suicidal substrates are not processed by TDP2 unless they first undergo proteolytic processing or denaturation. We also demonstrate that TDP2 is most efficient when the DNA attached to the tyrosyl is in a single-stranded configuration and that TDP2 can efficiently remove a tyrosine linked to a single misincorporated ribonucleotide or to polyribonucleotides, which expands the TDP2 catalytic profile with RNA substrates. The 1.6-Å resolution crystal structure of TDP2 bound to a substrate bearing a 5'-ribonucleotide defines a mechanism through which RNA can be accommodated in the TDP2 active site, albeit in a strained conformation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.565374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140274PMC
June 2014

Mapping topoisomerase sites in mitochondrial DNA with a poisonous mitochondrial topoisomerase I (Top1mt).

J Biol Chem 2014 Jun 5;289(26):18595-602. Epub 2014 May 5.

From the Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892

Mitochondrial topoisomerase I (Top1mt) is a type IB topoisomerase present in vertebrates and exclusively targeted to mitochondria. Top1mt relaxes mitochondrial DNA (mtDNA) supercoiling by introducing transient cleavage complexes wherein the broken DNA strand swivels around the intact strand. Top1mt cleavage complexes (Top1mtcc) can be stabilized in vitro by camptothecin (CPT). However, CPT does not trap Top1mtcc efficiently in cells and is highly cytotoxic due to nuclear Top1 targeting. To map Top1mtcc on mtDNA in vivo and to overcome the limitations of CPT, we designed two substitutions (T546A and N550H) in Top1mt to stabilize Top1mtcc. We refer to the double-mutant enzyme as Top1mt*. Using retroviral transduction and ChIP-on-chip assays with Top1mt* in Top1mt knock-out murine embryonic fibroblasts, we demonstrate that Top1mt* forms high levels of cleavage complexes preferentially in the noncoding regulatory region of mtDNA, accumulating especially at the heavy strand replication origin OH, in the ribosomal genes (12S and 16S) and at the light strand replication origin OL. Expression of Top1mt* also caused rapid mtDNA depletion without affecting mitochondria mass, suggesting the existence of specific mitochondrial pathways for the removal of damaged mtDNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.555367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140277PMC
June 2014

PARP1-TDP1 coupling for the repair of topoisomerase I-induced DNA damage.

Nucleic Acids Res 2014 Apr 3;42(7):4435-49. Epub 2014 Feb 3.

Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA, Laboratory of Molecular Biology, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India, Biotechnology and Cell Signaling, UMR7242 CNRS, Université de Strasbourg, Laboratory of Excellence Medalis, ESBS, Blvd Sébastien Brant, CS 10413, 67412 Illkirch, France, CEA-DSV-iMETI-SEPIA, BP6, 92265 Fontenay-aux-Roses cedex, France and Laboratory of Molecular Parasitology, Indian Institute of Chemical Biology, Kolkata 700032, India.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gku088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985661PMC
April 2014

Stereospecific PARP trapping by BMN 673 and comparison with olaparib and rucaparib.

Mol Cancer Ther 2014 Feb 19;13(2):433-43. Epub 2013 Dec 19.

Corresponding Author: Yves Pommier, Developmental Therapeutics Branch, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, 37 Convent Drive, Building 37, Room 5068, NIH, Bethesda, MD 20892-4255.

Anti-PARP drugs were initially developed as catalytic inhibitors to block the repair of DNA single-strand breaks. We recently reported that several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP-DNA complexes, and that both olaparib and niraparib act as PARP poisons at pharmacologic concentrations. Therefore, we have proposed that PARP inhibitors should be evaluated based both on catalytic PARP inhibition and PARP-DNA trapping. Here, we evaluated the novel PARP inhibitor, BMN 673, and compared its effects on PARP1 and PARP2 with two other clinical PARP inhibitors, olaparib and rucaparib, using biochemical and cellular assays in genetically modified chicken DT40 and human cancer cell lines. Although BMN 673, olaparib, and rucaparib are comparable at inhibiting PARP catalytic activity, BMN 673 is ∼100-fold more potent at trapping PARP-DNA complexes and more cytotoxic as single agent than olaparib, whereas olaparib and rucaparib show similar potencies in trapping PARP-DNA complexes. The high level of resistance of PARP1/2 knockout cells to BMN 673 demonstrates the selectivity of BMN 673 for PARP1/2. Moreover, we show that BMN 673 acts by stereospecific binding to PARP1 as its enantiomer, LT674, is several orders of magnitude less efficient. BMN 673 is also approximately 100-fold more cytotoxic than olaparib and rucaparib in combination with the DNA alkylating agents methyl methane sulfonate (MMS) and temozolomide. Our study demonstrates that BMN 673 is the most potent clinical PARP inhibitor tested to date with the highest efficiency at trapping PARP-DNA complexes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-13-0803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3946062PMC
February 2014

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs.

Nucleic Acids Res 2013 Sep 17;41(16):7793-803. Epub 2013 Jun 17.

Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA, Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan, NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, NIH, Rockville, MD 20850, USA and Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA.

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl-DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs-acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)-we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3'-ends. We also show that Tdp1-/- cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1-/- cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkt483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3763526PMC
September 2013

Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors.

Cancer Res 2012 Nov;72(21):5588-99

Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.

Small-molecule inhibitors of PARP are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single-strand breaks (SSB). However, the mechanism of action of PARP inhibitors with regard to their effects in cancer cells is not fully understood. In this study, we show that PARP inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA complexes were more cytotoxic than unrepaired SSBs caused by PARP inactivation, arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors with niraparib (MK-4827) > olaparib (AZD-2281) > veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP-DNA complexes using 30 genetically altered avian DT40 cell lines with preestablished deletions in specific DNA repair genes. This analysis revealed that, in addition to homologous recombination, postreplication repair, the Fanconi anemia pathway, polymerase β, and FEN1 are critical for repairing trapped PARP-DNA complexes. In summary, our study provides a new mechanistic foundation for the rational application of PARP inhibitors in cancer therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-12-2753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528345PMC
November 2012

Biochemical characterization of human tyrosyl-DNA phosphodiesterase 2 (TDP2/TTRAP): a Mg(2+)/Mn(2+)-dependent phosphodiesterase specific for the repair of topoisomerase cleavage complexes.

J Biol Chem 2012 Aug 20;287(36):30842-52. Epub 2012 Jul 20.

Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, MD 20892, USA.

TDP2 is a multifunctional enzyme previously known for its role in signal transduction as TRAF and TNF receptor-associated protein (TTRAP) and ETS1-associated protein 2 (EAPII). The gene has recently been renamed TDP2 because it plays a critical role for the repair of topoisomerase II cleavage complexes (Top2cc) and encodes an enzyme that hydrolyzes 5'-tyrosine-DNA adducts that mimic abortive Top2cc. Here we further elucidate the DNA-processing activities of human recombinant TDP2 and its biochemical characteristics. The preferred substrate for TDP2 is single-stranded DNA or duplex DNA with a four-base pair overhang, which is consistent with the known structure of Top2cc or Top3cc. The k(cat)/K(m) of TDP1 and TDP2 was determined. It was found to be 4 × 10(5) s(-1)M(-1) for TDP2 using single-stranded 5'-tyrosyl-DNA. The processing of substrates as short as five nucleotides long suggests that TDP2 can directly bind DNA ends. 5'-Phosphodiesterase activity requires a phosphotyrosyl linkage and tolerates an extended group attached to the tyrosine. TDP2 requires Mg(2+) or Mn(2+) for efficient catalysis but is weakly active with Ca(2+) or Zn(2+). Titration with Ca(2+) demonstrates a two-metal binding site in TDP2. Sequence alignment suggests that TDP2 contains four conserved catalytic motifs shared by Mg(2+)-dependent endonucleases, such as APE1. Substitutions at each of the four catalytic motifs identified key residues Asn-120, Glu-152, Asp-262, and His-351, whose mutation to alanine significantly reduced or completely abolished enzymatic activity. Our study characterizes the substrate specificity and kinetic parameters of TDP2. In addition, a two-metal catalytic mechanism is proposed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.393983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436327PMC
August 2012

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells.

J Biol Chem 2012 Apr 27;287(16):12848-57. Epub 2012 Feb 27.

Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.333963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3339927PMC
April 2012

Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) inhibitors.

Expert Opin Ther Pat 2011 Sep;21(9):1285-92

National Cancer Institute , Center for Cancer Research, Laboratory of Molecular Pharmacology , National Institutes of Health , Bethesda, MD, 20892 , USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1517/13543776.2011.604314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157054PMC
September 2011

Mutagenic processing of ribonucleotides in DNA by yeast topoisomerase I.

Science 2011 Jun;332(6037):1561-4

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.

The ribonuclease (RNase) H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nucleic acid metabolism. RNase H2 is specialized to remove single ribonucleotides [ribonucleoside monophosphates (rNMPs)] from duplex DNA, and its absence in budding yeast has been associated with the accumulation of deletions within short tandem repeats. Here, we demonstrate that rNMP-associated deletion formation requires the activity of Top1, a topoisomerase that relaxes supercoils by reversibly nicking duplex DNA. The reported studies extend the role of Top1 to include the processing of rNMPs in genomic DNA into irreversible single-strand breaks, an activity that can have distinct mutagenic consequences and may be relevant to human disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1205016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380281PMC
June 2011

The role of nucleotide cofactor binding in cooperativity and specificity of MutS recognition.

J Mol Biol 2008 Dec 27;384(1):31-47. Epub 2008 Aug 27.

Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, P.O. Box 208114, New Haven, CT 06520-8114, USA.

Mismatch repair (MMR) is essential for eliminating biosynthetic errors generated during replication or genetic recombination in virtually all organisms. The critical first step in Escherichia coli MMR is the specific recognition and binding of MutS to a heteroduplex, containing either a mismatch or an insertion/deletion loop of up to four nucleotides. All known MutS homologs recognize a similar broad spectrum of substrates. Binding and hydrolysis of nucleotide cofactors by the MutS-heteroduplex complex are required for downstream MMR activity, although the exact role of the nucleotide cofactors is less clear. Here, we showed that MutS bound to a 30-bp heteroduplex containing an unpaired T with a binding affinity approximately 400-fold stronger than to a 30-bp homoduplex, a much higher specificity than previously reported. The binding of nucleotide cofactors decreased both MutS specific and nonspecific binding affinity, with the latter marked by a larger drop, further increasing MutS specificity by approximately 3-fold. Kinetic studies showed that the difference in MutS K(d) for various heteroduplexes was attributable to the difference in intrinsic dissociation rate of a particular MutS-heteroduplex complex. Furthermore, the kinetic association event of MutS binding to heteroduplexes was marked by positive cooperativity. Our studies showed that the positive cooperativity in MutS binding was modulated by the binding of nucleotide cofactors. The binding of nucleotide cofactors transformed E. coli MutS tetramers, the functional unit in E. coli MMR, from a cooperative to a noncooperative binding form. Finally, we found that E. coli MutS bound to single-strand DNA with significant affinity, which could have important implication for strand discrimination in eukaryotic MMR mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2008.08.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2666446PMC
December 2008