APLF (C2orf13) is a novel component of poly(ADP-ribose) signaling in mammalian cells

APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly(ADP-ribose), a polymeric posttranslational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly(ADP-ribosyl)ation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly(ADP-ribosyl)ation in vitro, and this activity is dependent on its capacity to bind the polymer. In addition, transient overexpression in human A549 cells of full-length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly(ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly(ADP-ribose) and is a novel component of poly(ADP-ribose) signaling in mammalian cells

CDK targets Sae2 to control DNA-end resection and homologous recombination

DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR)1. HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template2-5. By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4​, ​6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs)2,3,5,7, with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2​, ​5). Here we establish that cell-cycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Δ null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection

The insecticides permethrin and chlorpyrifos show limited genotoxicity and no leukemogenic potential in human and murine hematopoietic stem progenitor cells

Altres ajuts: European Food and Safety Authority, EFSA.PRAS.2018.04-CT1; Consejo Nacional de Ciencia y Tecnología, CB-2012-01-183467; Centro de Investigación Biomédica en Red en Cáncer, PID2019-104695RBI00; Asociación Española Contra el Cáncer, AECC INVES211226MOLI

Effects of DNA and protein size on substrate cleavage by human tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyzes the hydrolysis of phosphodiester linkages between a DNA 3′ phosphate and a tyrosine residue as well as a variety of other DNA 3′ substituents, and has been implicated in the repair of covalent complexes involving eukaryotic type IB topoisomerases. To better understand the substrate features that are recognized by TDP1, the size of either the DNA or protein component of the substrate was varied. Competition experiments and gel shift analyses comparing a series of substrates with DNA lengths increasing from 6 to 28 nucleotides indicated that, contrary to predictions based on the crystal structure of the protein, the apparent affinity for the substrate increased as the DNA length was increased over the entire range tested. It has previously been found that a substrate containing the full-length native form of human topoisomerase I protein is not cleaved by TDP1. Protein-oligonucleotide complexes containing either a 53 or 108 amino acid long topoisomerase I-derived peptide were efficiently cleaved by TDP1, but like the full length protein, a substrate containing a 333 amino acid topoisomerase I fragment was resistant to cleavage. Consistent with these results, evidence is presented that processing by the proteasome is required for TDP1 cleavage in vivo

The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent of hemorrhagic colitis and the hemolytic uremic syndrome (HUS), produces long bundles of type IV pili (TFP) called hemorrhagic coli pili (HCP). HCP are capable of mediating several phenomena associated with pathogenicity: i) adherence to human and bovine epithelial cells; ii) invasion of epithelial cells; iii) hemagglutination of rabbit erythrocytes; iv) biofilm formation; v) twitching motility; and vi) specific binding to laminin and fibronectin. HCP are composed of a 19 kDa pilin subunit (HcpA) encoded by the hcpA chromosomal gene (called prepilin peptidase-dependent gene [ppdD] in E. coli K-12).In this study we investigated the potential role of HCP of E. coli O157:H7 strain EDL933 in activating the release of pro- and anti-inflammatory cytokines from a variety of host epithelial cells. We found that purified HCP and a recombinant HcpA protein induced significant release of IL-8 and TNF-alpha, from cultured polarized intestinal cells (T84 and HT-29 cells) and non-intestinal HeLa cells. Levels of proinflammatory IL-8 and TNF-alpha, but not IL-2, IL6, or IL-10 cytokines, were increased in the presence of HCP and recombinant HcpA after 6 h of incubation with >or=50 ng/ml of protein, suggesting that stimulation of IL-8 and TNF-alpha are dose and time-dependent. In addition, we also demonstrated that flagella are potent inducers of cytokine production. Furthermore, MAPK activation kinetics studies showed that EHEC induces p38 phosphorylation under HCP-producing conditions, and ERK1/2 and JNK activation was detectable after 3 h of EHEC infection. HT-29 cells were stimulated with epidermal growth factor stimulation of HT-29 cells for 30 min leading to activation of three MAPKs.The HcpA pilin monomer of the HCP produced by EHEC O157:H7 is a potent inducer of IL-8 and TNF-alpha release, an event which could play a significant role in the pathogenesis of hemorrhagic colitis caused by this pathogen

Mode of action of DNA-competitive small molecule inhibitors of tyrosyl DNA phosphodiesterase 2

TDP2 is a 5’-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II. TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for HTS-screening. We have gone on to determine crystal structures of these compounds bound to a ‘humanised’ form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2

MRE11 Function in Response to Topoisomerase Poisons Is Independent of its Function in Double-Strand Break Repair in Saccharomyces cerevisiae

Camptothecin (CPT) and etoposide (ETP) trap topoisomerase-DNA covalent intermediates, resulting in formation of DNA damage that can be cytotoxic if unrepaired. CPT and ETP are prototypes for molecules widely used in chemotherapy of cancer, so defining the mechanisms for repair of damage induced by treatment with these compounds is of great interest. In S. cerevisiae, deficiency in MRE11, which encodes a highly conserved factor, greatly enhances sensitivity to treatment with CPT or ETP. This has been thought to reflect the importance of double-strand break (DSB) repair pathways in the response to these to agents. Here we report that an S. cerevisiae strain expressing the mre11-H59A allele, mutant at a conserved active site histidine, is sensitive to hydroxyurea and also to ionizing radiation, which induces DSBs, but not to CPT or ETP. We show that TDP1, which encodes a tyrosyl-DNA phosphodiesterase activity able to release both 5′- and 3′-covalent topoisomerase-DNA complexes in vitro, contributes to ETP-resistance but not CPT-resistance in the mre11-H59A background. We further show that CPT- and ETP-resistance mediated by MRE11 is independent of SAE2, and thus independent of the coordinated functions of MRE11 and SAE2 in homology-directed repair and removal of Spo11 from DNA ends in meiosis. These results identify a function for MRE11 in the response to topoisomerase poisons that is distinct from its functions in DSB repair or meiotic DNA processing. They also establish that cellular proficiency in repair of DSBs may not correlate with resistance to topoisomerase poisons, a finding with potential implications for stratification of tumors with specific DNA repair deficiencies for treatment with these compounds

Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction

<p>Abstract</p> <p>Background</p> <p>Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg²⁺in its activity was not studied in sufficient details.</p> <p>Results</p> <p>In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg²⁺/Mn²⁺, and that neither Zn²⁺or nor Ca²⁺can activate hTDP2. Mg²⁺also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg²⁺controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg²⁺concentration above which it is inhibitory for hTDP2 activity.</p> <p>Conclusion</p> <p>These results altogether reveal the optimal Mg²⁺requirement in hTDP2 mediated reaction.</p