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
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
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
Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest, whereas a second barrier is mediated by oncogene-induced senescence. The relationship between these two barriers, if any, has not been elucidated. Here we show that oncogene-induced senescence is associated with signs of DNA replication stress, including prematurely terminated DNA replication forks and DNA double-strand breaks. Inhibiting the DNA double-strand break response kinase ataxia telangiectasia mutated (ATM) suppressed the induction of senescence and in a mouse model led to increased tumour size and invasiveness. Analysis of human precancerous lesions further indicated that DNA damage and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression.
- MeSH
- buněčné linie MeSH
- cyklin E genetika MeSH
- DNA MeSH
- geny mos MeSH
- inhibitor p16 cyklin-dependentní kinasy fyziologie MeSH
- invazivní růst nádoru genetika MeSH
- jaderné proteiny genetika MeSH
- lidé MeSH
- myši MeSH
- nádorová transformace buněk * genetika MeSH
- onkogeny * MeSH
- poškození DNA * MeSH
- prekancerózy genetika patologie MeSH
- proteiny buněčného cyklu genetika MeSH
- replikace DNA MeSH
- stárnutí buněk * genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
53BP1 is a key component of the genome surveillance network activated by DNA double strand breaks (DSBs). Despite its known accumulation at the DSB sites, the spatiotemporal aspects of 53BP1 interaction with DSBs and the role of other DSB regulators in this process remain unclear. Here, we used real-time microscopy to study the DSB-induced redistribution of 53BP1 in living cells. We show that within minutes after DNA damage, 53BP1 becomes progressively, yet transiently, immobilized around the DSB-flanking chromatin. Quantitative imaging of single cells revealed that the assembly of 53BP1 at DSBs significantly lagged behind Mdc1/NFBD1, another DSB-interacting checkpoint mediator. Furthermore, short interfering RNA-mediated ablation of Mdc1/NFBD1 drastically impaired 53BP1 redistribution to DSBs and triggered premature dissociation of 53BP1 from these regions. Collectively, these in vivo measurements identify Mdc1/NFBD1 as a key upstream determinant of 53BP1's interaction with DSBs from its dynamic assembly at the DSB sites through sustained retention within the DSB-flanking chromatin up to the recovery from the checkpoint.
- MeSH
- chromatin fyziologie MeSH
- DNA vazebné proteiny fyziologie genetika MeSH
- fosfoproteiny fyziologie genetika MeSH
- intracelulární signální peptidy a proteiny fyziologie genetika MeSH
- jaderné proteiny fyziologie genetika MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- malá interferující RNA fyziologie MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- poškození DNA * MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- trans-aktivátory fyziologie genetika MeSH
- transport proteinů MeSH
- vazba proteinů MeSH
- zelené fluorescenční proteiny genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
