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

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

The protein kinases ataxia-telangiectasia mutated (ATM) and ATM-Rad3 related (ATR) are activated in response to DNA damage, genotoxic stress and virus infections. Here we show that during infection with wild-type adenovirus, ATR and its cofactors RPA32, ATRIP and TopBP1 accumulate at viral replication centres, but there is minimal ATR activation. We show that the Mre11/Rad50/Nbs1 (MRN) complex is recruited to viral centres only during infection with adenoviruses lacking the early region E4 and ATR signaling is activated. This suggests a novel requirement for the MRN complex in ATR activation during virus infection, which is independent of Mre11 nuclease activity and recruitment of RPA/ATR/ATRIP/TopBP1. Unlike other damage scenarios, we found that ATM and ATR signaling are not dependent on each other during infection. We identify a region of the viral E4orf3 protein responsible for immobilization of the MRN complex and show that this prevents ATR signaling during adenovirus infection. We propose that immobilization of the MRN damage sensor by E4orf3 protein prevents recognition of viral genomes and blocks detrimental aspects of checkpoint signaling during virus infection.

• MeSH
• Adenoviridae MeSH
• adenovirové infekce * metabolismus   MeSH
• adenovirové proteiny E4 chemie   metabolismus   MeSH
• ATM protein MeSH
• buněčné linie MeSH
• DNA vazebné proteiny * metabolismus   MeSH
• enzymy opravy DNA * metabolismus   MeSH
• fosforylace MeSH
• jaderné proteiny * metabolismus   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• multiproteinové komplexy * metabolismus   MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• protein-serin-threoninkinasy * metabolismus   MeSH
• proteiny buněčného cyklu * metabolismus   MeSH
• replikace viru MeSH
• sekvence aminokyselin MeSH
• signální transdukce * MeSH
• transport proteinů MeSH
• Check Tag
• lidé MeSH

Claspin is an adaptor protein that facilitates the ataxia telangiectasia and Rad3-related (ATR)-mediated phosphorylation and activation of Chk1, a key effector kinase in the DNA damage response. Efficient termination of Chk1 signaling in mitosis and during checkpoint recovery requires SCF(betaTrCP)-dependent destruction of Claspin. Here, we identify the deubiquitylating enzyme ubiquitin-specific protease 7 (USP7) as a novel regulator of Claspin stability. Claspin and USP7 interact in vivo, and USP7 is required to maintain steady-state levels of Claspin. Furthermore, USP7-mediated deubiquitylation markedly prolongs the half-life of Claspin, which in turn increases the magnitude and duration of Chk1 phosphorylation in response to genotoxic stress. Finally, we find that in addition to the M phase-specific, SCF(betaTrCP)-mediated degradation, Claspin is destabilized by the anaphase-promoting complex (APC) and thus remains unstable in G1. Importantly, we demonstrate that USP7 specifically opposes the SCF(betaTrCP)- but not APC(Cdh1)-mediated degradation of Claspin. Thus, Claspin turnover is controlled by multiple ubiquitylation and deubiquitylation activities, which together provide a flexible means to regulate the ATR-Chk1 pathway.

• MeSH
• adaptorové proteiny signální transdukční * metabolismus   MeSH
• anafázi podporující komplex MeSH
• buněčné linie MeSH
• fosforylace MeSH
• G1 fáze MeSH
• komplexy ubikvitinligas * metabolismus   fyziologie   MeSH
• lidé MeSH
• poškození DNA MeSH
• proteinkinasy metabolismus   MeSH
• proteinligasy komplexu SCF * metabolismus   fyziologie   MeSH
• substrátová specifita MeSH
• thiolesterasa ubikvitinu * metabolismus   fyziologie   MeSH
• ubikvitinace fyziologie   MeSH
• Check Tag
• lidé MeSH

DNA double-strand breaks (DSBs) trigger accumulation of the MRE11-RAD50-Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex, whose retention on the DSB-flanking chromatin facilitates survival. Chromatin retention of MRN requires the MDC1 adaptor protein, but the mechanism behind the MRN-MDC1 interaction is unknown. We show that the NBS1 subunit of MRN interacts with the MDC1 N terminus enriched in Ser-Asp-Thr (SDT) repeats. This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1. Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1-NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells. Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs. Together, these data suggest that phosphorylation of the SDT repeats in the MDC1 N terminus functions to recruit NBS1 and, thereby, increases the local concentration of MRN at the sites of chromosomal breakage.

• MeSH
• DNA nádorová genetika   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• fosforylace MeSH
• imunohistochemie MeSH
• jaderné proteiny * genetika   metabolismus   MeSH
• klonování DNA MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory kostí MeSH
• oligopeptidy * metabolismus   MeSH
• osteosarkom MeSH
• peptidové fragmenty metabolismus   MeSH
• polymerázová řetězová reakce MeSH
• poškození DNA * MeSH
• proteiny buněčného cyklu * genetika   metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• trans-aktivátory * genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH

DNA strand breaks arise continuously as the result of intracellular metabolism and in response to a multitude of genotoxic agents. To overcome such challenges to genomic stability, cells have evolved genome surveillance pathways that detect and repair damaged DNA in a coordinated fashion. Here we identify the previously uncharacterized human protein Xip1 (C2orf13) as a novel component of the checkpoint response to DNA strand breaks. Green fluorescent protein-tagged Xip1 was rapidly recruited to sites of DNA breaks, and this accumulation was dependent on a novel type of zinc finger motif located in the C terminus of Xip1. The initial recruitment kinetics of Xip1 closely paralleled that of XRCC1, a central organizer of single strand break (SSB) repair, and its accumulation was both delayed and sustained when the detection of SSBs was abrogated by inhibition of PARP-1. Xip1 and XRCC1 stably interacted through recognition of CK2 phosphorylation sites in XRCC1 by the Forkhead-associated (FHA) domain of Xip1, and XRCC1 was required to maintain steady-state levels of Xip1. Moreover, Xip1 was phosphorylated on Ser-116 by ataxia telangiectasia-mutated in response to ionizing radiation, further underscoring the potential importance of Xip1 in the DNA damage response. Finally, depletion of Xip1 significantly decreased the clonogenic survival of cells exposed to DNA SSB- or double strand break-inducing agents. Collectively, these findings implicate Xip1 as a new regulator of genome maintenance pathways, which may function to organize DNA strand break repair complexes at sites of DNA damage.

• MeSH
• ATM protein MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-lyasa (apurinová nebo apyrimidinová) MeSH
• dvouřetězcové zlomy DNA * MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• fosforylace MeSH
• jednořetězcové zlomy DNA * MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nestabilita genomu * fyziologie   MeSH
• oprava DNA * fyziologie   MeSH
• posttranslační úpravy proteinů fyziologie   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• terciární struktura proteinů genetika   MeSH
• zinkové prsty genetika   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

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

We show that Claspin, an adaptor protein required for Chk1 activation, becomes degraded at the onset of mitosis. Claspin degradation was triggered by its interaction with, and ubiquitylation by, the SCFbetaTrCP ubiquitin ligase. This interaction was phosphorylation dependent and required the activity of the Plk1 kinase and the integrity of a betaTrCP recognition motif (phosphodegron) in the N terminus of Claspin. Uncoupling of Claspin from betaTrCP by mutating the conserved serines in Claspin's phosphodegron or by knocking down betaTrCP stabilized Claspin in mitosis, impaired Chk1 dephosphorylation, and delayed G2/M transition during recovery from cell cycle arrest imposed by DNA damage or replication stress. Moreover, the inability to degrade Claspin allowed partial reactivation of Chk1 in cells exposed to DNA damage after passing the G2/M transition. Our data suggest that degradation of Claspin facilitates timely reversal of the checkpoint response and delineates the period permissive for Chk1 activation during cell cycle progression.

• MeSH
• adaptorové proteiny signální transdukční fyziologie   genetika   MeSH
• biologické modely MeSH
• buněčné linie MeSH
• buněčný cyklus účinky léků   MeSH
• cyklin A genetika   metabolismus   MeSH
• daunomycin farmakologie   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fosforylace účinky léků   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• malá interferující RNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• nádorové buněčné linie MeSH
• poškození DNA * fyziologie   MeSH
• proteinkinasy * genetika   metabolismus   MeSH
• proteinligasy komplexu SCF genetika   metabolismus   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• transfekce MeSH
• tyrosinkinasy genetika   metabolismus   MeSH
• ubikvitin metabolismus   MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH