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

Following the discovery in 1998 of -H2AX, the first histone modification induced by DNA damage, interest in the changes to chromatin induced by DNA damage has exploded, and a vast amount of information has been generated. However, there has been a discrepancy between our rapidly advancing knowledge of how chromatin responds to DNA damage and the understanding of why cells mobilize large segments of chromatin to protect the genome against destabilizing effects posed by tiny DNA lesions. Recent research has provided insights into these issues and suggests that chromatin responses induced by DNA damage are not simply the accumulation of 'nuclear foci' but are mechanisms required to guard genome integrity.

• MeSH
• buněčný cyklus * genetika   MeSH
• histony * metabolismus   MeSH
• lidé MeSH
• nestabilita genomu * MeSH
• oprava DNA MeSH
• poškození DNA * MeSH
• posttranslační úpravy proteinů MeSH
• restrukturace chromatinu * genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH

Maintaining genomic integrity is critical to avoid life-threatening disorders, such as premature aging, neurodegeneration and cancer. A multiprotein cascade operates at sites of DNA double-strand breaks (DSBs) to recognize, signal and repair damage. RNF168 (ring-finger nuclear factor) contributes to this emerging pathway of several E3 ubiquitin ligases that perform sequential ubiquitylations on damaged chromosomes, chromatin modifications essential for aggregation of repair complexes at the DSB sites. Here, we report the clinical and cellular phenotypes associated with a newly identified homozygous nonsense mutation in the RNF168 gene of a patient with a syndrome mimicking ataxia-telangiectasia. The mutation eliminated both of RNF168's ubiquitin-binding motifs, thus blocking progression of the ubiquitylation cascade and retention of repair proteins including tumor suppressors 53BP1 and BRCA1 at DSB sites, consistent with the observed defective DNA damage checkpoints/repair and pronounced radiosensitivity. Rapid screening for RNF168 pathway deficiency was achieved by scoring patients' lymphoblastoid cells for irradiation-induced nuclear foci containing 53BP1, a robust assay we propose for future diagnostic applications. The formation of radiation-induced DSB repair foci was rescued by ectopic expression of wild-type RNF168 in patient's cells, further causally linking the RNF168 mutation with the pathology. Clinically, this novel syndrome featured ataxia, telangiectasia, elevated alphafetoprotein, immunodeficiency, microcephaly and pulmonary failure and has implications for the differential diagnosis of autosomal recessive ataxias.

• MeSH
• diferenciální diagnóza MeSH
• homozygot MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• lidé MeSH
• mladiství MeSH
• nesmyslný kodon * MeSH
• poruchy růstu genetika   parazitologie   MeSH
• protein BRCA1 genetika   metabolismus   MeSH
• syndrom MeSH
• teleangiektatická ataxie * diagnóza   genetika   metabolismus   patofyziologie   MeSH
• tolerance záření genetika   MeSH
• ubikvitin genetika   metabolismus   MeSH
• ubikvitinligasy * nedostatek   genetika   MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mužské pohlaví 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

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate-dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21(Cip1) accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.

• MeSH
• autoantigeny genetika   metabolismus   MeSH
• buněčný cyklus genetika   MeSH
• CDC geny MeSH
• chromatin * genetika   metabolismus   MeSH
• chromozomy metabolismus   MeSH
• DNA genetika   metabolismus   MeSH
• dvouřetězcové zlomy DNA MeSH
• fosfatasy cdc25 genetika   metabolismus   MeSH
• ionizující záření MeSH
• komplex Mi2-NuRD genetika   metabolismus   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   farmakologie   MeSH
• nádorové buněčné linie MeSH
• oprava DNA * MeSH
• poškození DNA * fyziologie   MeSH
• RNA interference MeSH
• signální transdukce * genetika   MeSH
• ubikvitin genetika   metabolismus   MeSH
• ubikvitinace MeSH
• Check Tag
• lidé MeSH

DNA double-strand breaks (DSBs) not only interrupt the genetic information, but also disrupt the chromatin structure, and both impairments require repair mechanisms to ensure genome integrity. We showed previously that RNF8-mediated chromatin ubiquitylation protects genome integrity by promoting the accumulation of repair factors at DSBs. Here, we provide evidence that, while RNF8 is necessary to trigger the DSB-associated ubiquitylations, it is not sufficient to sustain conjugated ubiquitin in this compartment. We identified RNF168 as a novel chromatin-associated ubiquitin ligase with an ability to bind ubiquitin. We show that RNF168 interacts with ubiquitylated H2A, assembles at DSBs in an RNF8-dependent manner, and, by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. Thus, RNF168 defines a new pathway involving sequential ubiquitylations on damaged chromosomes and uncovers a functional cooperation between E3 ligases in genome maintenance.

• MeSH
• buněčné linie MeSH
• chromozomy * metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• dvouřetězcové zlomy DNA * MeSH
• genový knockdown MeSH
• histony metabolismus   MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• lidé MeSH
• oprava DNA * MeSH
• terciární struktura proteinů MeSH
• ubikvitin * metabolismus   MeSH
• ubikvitinligasy * chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH

Accumulation of repair proteins on damaged chromosomes is required to restore genomic integrity. However, the mechanisms of protein retention at the most destructive chromosomal lesions, the DNA double-strand breaks (DSBs), are poorly understood. We show that RNF8, a RING-finger ubiquitin ligase, rapidly assembles at DSBs via interaction of its FHA domain with the phosphorylated adaptor protein MDC1. This is accompanied by an increase in DSB-associated ubiquitylations and followed by accumulation of 53BP1 and BRCA1 repair proteins. Knockdown of RNF8 or disruption of its FHA or RING domains impaired DSB-associated ubiquitylation and inhibited retention of 53BP1 and BRCA1 at the DSB sites. In addition, we show that RNF8 can ubiquitylate histone H2A and H2AX, and that its depletion sensitizes cells to ionizing radiation. These data suggest that MDC1-mediated and RNF8-executed histone ubiquitylation protects genome integrity by licensing the DSB-flanking chromatin to concentrate repair factors near the DNA lesions.

• MeSH
• biologické modely MeSH
• chromatin metabolismus   MeSH
• DNA vazebné proteiny fyziologie   metabolismus   MeSH
• dvouřetězcové zlomy DNA * MeSH
• enzymy opravy DNA * metabolismus   MeSH
• fosforylace MeSH
• histony * metabolismus   MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• jaderné proteiny metabolismus   MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• protein BRCA1 metabolismus   MeSH
• terciární struktura proteinů MeSH
• trans-aktivátory metabolismus   MeSH
• ubikvitinace * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH

In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient homologous recombination. Finally, we reveal that CtIP has sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination.

• MeSH
• ATM protein MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA * metabolismus   MeSH
• dvouřetězcové zlomy DNA účinky léků   MeSH
• endonukleasy MeSH
• G2 fáze MeSH
• jaderné proteiny genetika   metabolismus   nedostatek   MeSH
• jednovláknová DNA metabolismus   MeSH
• konzervovaná sekvence MeSH
• lidé MeSH
• molekulární evoluce MeSH
• nádorové buněčné linie MeSH
• oprava DNA * účinky léků   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• rekombinace genetická * účinky léků   MeSH
• S fáze MeSH
• Saccharomyces cerevisiae - proteiny chemie   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH

We show that DNA double-strand breaks (DSBs) induce complex subcompartmentalization of genome surveillance regulators. Chromatin marked by gamma-H2AX is occupied by ataxia telangiectasia-mutated (ATM) kinase, Mdc1, and 53BP1. In contrast, repair factors (Rad51, Rad52, BRCA2, and FANCD2), ATM and Rad-3-related (ATR) cascade (ATR, ATR interacting protein, and replication protein A), and the DNA clamp (Rad17 and -9) accumulate in subchromatin microcompartments delineated by single-stranded DNA (ssDNA). BRCA1 and the Mre11-Rad50-Nbs1 complex interact with both of these compartments. Importantly, some core DSB regulators do not form cytologically discernible foci. These are further subclassified to proteins that connect DSBs with the rest of the nucleus (Chk1 and -2), that assemble at unprocessed DSBs (DNA-PK/Ku70), and that exist on chromatin as preassembled complexes but become locally modified after DNA damage (Smc1/Smc3). Finally, checkpoint effectors such as p53 and Cdc25A do not accumulate at DSBs at all. We propose that subclassification of DSB regulators according to their residence sites provides a useful framework for understanding their involvement in diverse processes of genome surveillance.

• MeSH
• buněčné linie MeSH
• chromatin fyziologie   MeSH
• chromozomální proteiny, nehistonové fyziologie   MeSH
• DNA * metabolismus   účinky záření   MeSH
• fosforylace MeSH
• genom * MeSH
• jaderné proteiny fyziologie   MeSH
• kultivované buňky MeSH
• lasery MeSH
• lidé MeSH
• oprava DNA * MeSH
• poškození DNA * MeSH
• protein BRCA1 fyziologie   MeSH
• proteinkinasy fyziologie   MeSH
• proteiny buněčného cyklu * fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH

It is generally thought that the DNA-damage checkpoint kinases, ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), work independently of one another. Here, we show that ATM and the nuclease activity of meiotic recombination 11 (Mre11) are required for the processing of DNA double-strand breaks (DSBs) to generate the replication protein A (RPA)-coated ssDNA that is needed for ATR recruitment and the subsequent phosphorylation and activation of Chk1. Moreover, we show that efficient ATM-dependent ATR activation in response to DSBs is restricted to the S and G2 cell cycle phases and requires CDK kinase activity. Thus, in response to DSBs, ATR activation is regulated by ATM in a cell-cycle dependent manner.

• MeSH
• ATM protein MeSH
• buněčné jádro metabolismus   MeSH
• buněčný cyklus * MeSH
• cyklin-dependentní kinasy metabolismus   MeSH
• DNA vazebné proteiny fyziologie   chemie   MeSH
• fosforylace MeSH
• HeLa buňky MeSH
• jaderné proteiny chemie   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové supresorové proteiny fyziologie   chemie   MeSH
• poškození DNA * MeSH
• protein-serin-threoninkinasy fyziologie   chemie   metabolismus   MeSH
• proteinkinasy metabolismus   MeSH
• proteiny buněčného cyklu fyziologie   chemie   metabolismus   MeSH
• replikační protein A chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH