To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometry-based proteomics to measure site-specific phosphorylation changes of nuclear proteins after ionizing radiation. We profiled 5204 phosphorylation sites at five time points following DNA damage of which 594 sites on 209 proteins were observed to be regulated more than 2-fold. Of the 594 sites, 372 are novel phosphorylation sites primarily of nuclear origin. The 594 sites could be classified to distinct temporal profiles. Sites regulated shortly after radiation were enriched in the ataxia telangiectasia mutated (ATM) kinase SQ consensus sequence motif and a novel SXXQ motif. Importantly, in addition to induced phosphorylation, we identified a considerable group of sites that undergo DNA damage-induced dephosphorylation. Together, our data extend the number of known phosphorylation sites regulated by DNA damage, provides so far unprecedented temporal dissection of DNA damage-modified phosphorylation events, and elucidate the cross-talk between different types of post-translational modifications in the dynamic regulation of a multifaceted DNA damage response.

• MeSH
• aminokyselinové motivy MeSH
• buněčné jádro * metabolismus   MeSH
• buněčné linie MeSH
• časové faktory MeSH
• chromatografie MeSH
• fosforylace MeSH
• jaderné proteiny * chemie   metabolismus   MeSH
• konsenzuální sekvence MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• poškození DNA * MeSH
• posttranslační úpravy proteinů MeSH
• proteinkinasy chemie   metabolismus   MeSH
• proteom * chemie   metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• sekvence aminokyselin MeSH
• shluková analýza MeSH
• signální transdukce 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

ATR is a protein kinase that orchestrates the cellular response to replication problems and DNA damage. HCLK2 has previously been reported to stabilize ATR and Chk1. Here we provide evidence that human HCLK2 acts at an early step in the ATR signaling pathway and contributes to full-scale activation of ATR kinase activity. We show that HCLK2 forms a complex with ATR-ATRIP and the ATR activator TopBP1. We demonstrate that HCLK2-induced ATR kinase activity toward substrates requires TopBP1 and vice versa and provides evidence that HCLK2 facilitates efficient ATR-TopBP1 association. Consistent with its role in ATR activation, HCLK2 depletion severely impaired phosphorylation of multiple ATR targets including Chk1, Nbs1, and Smc1 after DNA damage. We show that HCLK2 is required for and stimulates ATR autophosphorylation and activity toward different substrates in vitro. Furthermore, HCLK2 depletion abrogated the G(2) checkpoint and decreased survival of cells after exposure to DNA damaging agents and replicative stress. Overall, our data suggest that HCLK2 facilitates ATR activation and, therefore, contributes to ATR-mediated checkpoint signaling. Importantly, our results suggest that HCLK2 functions in the same pathway as TopBP1 but that the two proteins regulate different steps in ATR activation.

• MeSH
• ATM protein MeSH
• chromozomální proteiny, nehistonové genetika   metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• enzymová indukce fyziologie   MeSH
• HeLa buňky MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• poškození DNA * fyziologie   MeSH
• protein-serin-threoninkinasy * genetika   metabolismus   MeSH
• proteinkinasy genetika   metabolismus   MeSH
• proteiny buněčného cyklu * genetika   metabolismus   MeSH
• stabilita enzymů fyziologie   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• tyrosinkinasy * genetika   metabolismus   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