53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-β interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-β pathway and implicate this mechanism in the maintenance of genome integrity.

• MeSH
• buněčné jádro metabolismus   MeSH
• genom lidský genetika   MeSH
• HeLa buňky MeSH
• imunoblotting MeSH
• imunoprecipitace MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• komplex proteinů jaderného póru genetika   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• RNA interference fyziologie   MeSH
• vazba proteinů genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis. Copyright 2012 Elsevier Inc. All rights reserved.

• MeSH
• Alphapapillomavirus MeSH
• buněčné linie MeSH
• chromatin * metabolismus   MeSH
• dvouřetězcové zlomy DNA * MeSH
• genetická transkripce MeSH
• infekce papilomavirem metabolismus   patologie   MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory metabolismus   patologie   virologie   MeSH
• oprava DNA * MeSH
• transportní proteiny * metabolismus   MeSH
• ubikvitinace MeSH
• ubikvitinligasy * metabolismus   MeSH
• umlčování genů MeSH
• Check Tag
• lidé MeSH

Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication stress increases the frequency of chromosomal lesions that are transmitted to daughter cells. Throughout G1, these lesions are sequestered in nuclear compartments marked by p53-binding protein 1 (53BP1) and other chromatin-associated genome caretakers. We show that the number of such 53BP1 nuclear bodies increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations. 2011 Macmillan Publishers Limited. All rights reserved.

• MeSH
• buněčné jádro * metabolismus   MeSH
• buněčný cyklus MeSH
• časové faktory MeSH
• chromatin metabolismus   MeSH
• chromozomy * ultrastruktura   MeSH
• DNA-helikasy metabolismus   MeSH
• DNA * genetika   metabolismus   MeSH
• intracelulární signální peptidy a proteiny * metabolismus   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• mitóza * MeSH
• nádorové buněčné linie MeSH
• poškození DNA MeSH
• regulace genové exprese u nádorů MeSH
• replikace DNA * MeSH
• Check Tag
• lidé MeSH

• Publikační typ
• abstrakt z konference MeSH

• Klíčová slova
• škodlivé působení aerosolu CrO3 na pracující při galvanizaci,
• MeSH
• aspirace do dýchacích cest patologie   prevence a kontrola   MeSH
• biomedicínský výzkum MeSH
• lidé MeSH
• nemoci z povolání * diagnóza   etiologie   chemicky indukované   MeSH
• otorinolaryngologické nádory * diagnóza   etiologie   MeSH
• pracovní lékařství metody   MeSH
• sloučeniny chromu * škodlivé účinky   toxicita   MeSH
• statistika jako téma MeSH
• Check Tag
• lidé MeSH