Suchergebnisse

Intramural research program of the US National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS) [1Z01ES102765 to R.S.W.]; Work in the F.C.L. lab is supported by Ministerio de Economía y Competitividad, Gobierno de España [SAF2017-89619-R, European Regional Development Fund]; European Research Council [ERC-CoG-2014-647359]; University of Seville Predoctoral Studentship [PIF-2011 to J.A.L.]; M.J.S. is supported by Mayo Clinic start-up funds and the Center for Biomedical Discovery new investigator funds; Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory; Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [W-31-109-Eng-38]; SAXS data were collected at the Advanced Light Source (ALS), a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program, supported by DOE Office of Biological and Environmental Research. Additional support comes from the National Institute of Health project ALS-ENABLE [P30 GM124169]; High-End Instrumentation Grant [S10OD018483]. Funding for open access charge: US government, Intramural NIH. ; Tyrosyl-DNA phosphodiesterase 2 (TDP2) reverses Topoisomerase 2 DNA-protein crosslinks (TOP2-DPCs) in a direct-reversal pathway licensed by ZATTZNF451 SUMO2 E3 ligase and SUMOylation of TOP2. TDP2 also binds ubiquitin (Ub), but how Ub regulates TDP2 functions is unknown. Here, we show that TDP2 co-purifies with K63 and K27 poly-Ubiquitinated cellular proteins independently of, and separately from SUMOylated TOP2 complexes. Poly-ubiquitin chains of ≥ Ub3 stimulate TDP2 catalytic activity in nuclear extracts and enhance TDP2 binding of DNA-protein crosslinks in vitro. X-ray crystal structures and small-angle X-ray scattering analysis of TDP2-Ub complexes reveal that the TDP2 UBA domain binds K63-Ub3 in a 1:1 stoichiometric complex that relieves a UBA-regulated autoinhibitory state of TDP2. Our data indicates that that poly-Ub regulates TDP2-catalyzed TOP2-DPC removal, and TDP2 single nucleotide polymorphisms can disrupt the TDP2-Ubiquitin interface. ; Sí

Tyrosyl-DNA phosphodiesterase 2 (TDP2) reverses Topoisomerase 2 DNA–protein crosslinks (TOP2-DPCs) in a direct-reversal pathway licensed by ZATTZNF451 SUMO2 E3 ligase and SUMOylation of TOP2. TDP2 also binds ubiquitin (Ub), but how Ub regulates TDP2 functions is unknown. Here, we show that TDP2 co-purifies with K63 and K27 poly-Ubiquitinated cellular proteins independently of, and separately from SUMOylated TOP2 complexes. Poly-ubiquitin chains of ≥ Ub3 stimulate TDP2 catalytic activity in nuclear extracts and enhance TDP2 binding of DNA–protein crosslinks in vitro. X-ray crystal structures and small-angle X-ray scattering analysis of TDP2-Ub complexes reveal that the TDP2 UBA domain binds K63-Ub3 in a 1:1 stoichiometric complex that relieves a UBA-regulated autoinhibitory state of TDP2. Our data indicates that that poly-Ub regulates TDP2-catalyzed TOP2-DPC removal, and TDP2 single nucleotide polymorphisms can disrupt the TDP2-Ubiquitin interface. ; Intramural research program of the US National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS) [1Z01ES102765 to R.S.W.]; Work in the F.C.L. lab is supported by Ministerio de Economía y Competitividad, Gobierno de España [SAF2017-89619-R, European Regional Development Fund]; European Research Council [ERC-CoG-2014-647359]; University of Seville Predoctoral Studentship [PIF-2011 to J.A.L.]; M.J.S. is supported by Mayo Clinic start-up funds and the Center for Biomedical Discovery new investigator funds; Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory; Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [W-31-109-Eng-38]; SAXS data were collected at the Advanced Light Source (ALS), a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program, supported by DOE Office of Biological and Environmental Research. Additional support comes from the National Institute of Health project ALS-ENABLE [P30 GM124169]; High-End Instrumentation Grant [S10OD018483]. Funding for open access charge: US government, Intramural NIH. ; Peer reviewed

Versión postprint próximamente disponible en: http://hdl.handle.net/10261/228202 ; The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 28 CRISPR/Cas9 screens against 25 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 840 genes whose loss causes either sensitivity or resistance to DNA damaging agents. Mining this dataset, we uncovered that ERCC6L2, which is mutated in a bone-marrow failure syndrome, codes for a canonical non-homologous end-joining pathway factor; that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy. ; AAQ, GSM and AMH are recipients of long-term EMBO fellowships (ALTF 910-2017, 795-2017 under a CC-BY-NC-ND 4.0 International license. not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available bioRxiv preprint doi: https://doi.org/10.1101/845446; this version posted November 18, 2019. The copyright holder for this preprint (which was 27 and 124-2019, respectively), NH was supported by a Human Frontier Science Program long-term Fellowship, SER is supported by a fellowship from AIRC and SA was a Banting post-doctoral fellow. ASB was supported by a PhD Studentship from AEFAT (Asociación Española Familia Ataxia Telangiectasia) and an EMBO short-term fellowship for a visit to the DD laboratory. The ICRF187 screen in FCL laboratory was funded by grants from the Spanish Government (SAF2017- 89619-R, European Regional Development Fund) and the European Research Council (ERC-CoG2014-647359). Work in the RSW laboratory was supported in part by the US National Institute of Health Intramural Program, US National Institute of Environmental Health Sciences (NIEHS, 1Z01ES102765). DD is a Canada Research Chair (Tier I) and the work was supported from grants from the CIHR (FDN143343 to DD; FRN 123518, PJT-156330 to AM) Canadian Cancer Society grant 705644 (to DD) with additional support to DD from the Krembil Foundation. ; No