The conserved MRE11-RAD50-NBS1 (MRN) complex is an important sensor of DNA double-strand breaks (DSBs) and facilitates DNA repair by homologous recombination (HR) and end joining. Here, we identify NBS1 as a target of cyclin-dependent kinase (CDK) phosphorylation. We show that NBS1 serine 432 phosphorylation occurs in the S, G2 and M phases of the cell cycle and requires CDK activity. This modification stimulates MRN-dependent conversion of DSBs into structures that are substrates for repair by HR. Impairment of NBS1 phosphorylation not only negatively affects DSB repair by HR, but also prevents resumption of DNA replication after replication-fork stalling. Thus, CDK-mediated NBS1 phosphorylation defines a molecular switch that controls the choice of repair mode for DSBs.

• MeSH
• DNA vazebné proteiny chemie   MeSH
• dvouřetězcové zlomy DNA MeSH
• enzymy opravy DNA chemie   MeSH
• homologní rekombinace * MeSH
• jaderné proteiny * genetika   metabolismus   MeSH
• lidé MeSH
• mutageneze cílená MeSH
• nádorové buněčné linie MeSH
• oprava DNA MeSH
• proteinkinasa CDC2 * chemie   metabolismus   MeSH
• proteiny buněčného cyklu * chemie   genetika   metabolismus   MeSH
• replikace DNA * MeSH
• štěpení DNA * MeSH
• substituce aminokyselin MeSH
• Check Tag
• lidé MeSH

The prime objective for every life form is to deliver its genetic material, intact and unchanged, to the next generation. This must be achieved despite constant assaults by endogenous and environmental agents on the DNA. To counter this threat, life has evolved several systems to detect DNA damage, signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management.

• MeSH
• buněčný cyklus fyziologie   MeSH
• genom lidský genetika   MeSH
• lidé MeSH
• nemoc * MeSH
• oprava DNA * genetika   fyziologie   MeSH
• poškození DNA * genetika   fyziologie   MeSH
• signální transdukce 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

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

Mdc1/NFBD1 controls cellular responses to DNA damage, in part via interacting with the Mre11-Rad50-Nbs1 complex that is involved in the recognition, signalling, and repair of DNA double-strand breaks (DSBs). Here, we show that in live human cells, the transient interaction of Nbs1 with DSBs and its phosphorylation by ATM are Mdc1-independent. However, ablation of Mdc1 by siRNA or mutation of the Nbs1's FHA domain required for Mdc1 binding reduced the affinity of Nbs1 for DSB-flanking chromatin and caused aberrant pan-nuclear dispersal of Nbs1. This occurred despite normal phosphorylation of H2AX, indicating that lack of Mdc1 does not impair this DSB-induced chromatin change, but rather precludes the sustained engagement of Nbs1 with these regions. Mdc1 (but not Nbs1) became partially immobilized to chromatin after DSB generation, and siRNA-mediated depletion of H2AX prevented such relocalization of Mdc1 and uncoupled Nbs1 from DSB-flanking chromatin. Our data suggest that Mdc1 functions as an H2AX-dependent interaction platform enabling a switch from transient, Mdc1-independent recruitment of Nbs1 to DSBs towards sustained, Mdc1-dependent interactions with the surrounding chromosomal microenvironment.

• MeSH
• buněčné jádro metabolismus   MeSH
• chromatin * metabolismus   MeSH
• DNA vazebné proteiny * metabolismus   MeSH
• fluorescenční barviva MeSH
• fluorescenční protilátková technika MeSH
• fosfoproteiny metabolismus   MeSH
• histony * metabolismus   MeSH
• hydraziny MeSH
• jaderné proteiny chemie   metabolismus   MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• monoklonální protilátky MeSH
• nádorové buněčné linie MeSH
• osteosarkom patologie   MeSH
• poškození DNA * MeSH
• proteiny buněčného cyklu chemie   metabolismus   MeSH
• RNA interference MeSH
• terciární struktura proteinů MeSH
• trans-aktivátory * MeSH
• Check Tag
• lidé MeSH

MRE11, RAD50 and NBS1 form a highly conserved protein complex (the MRE11 complex) that is involved in the detection, signalling and repair of DNA damage. We identify MDC1 (KIAA0170/NFBD1), a protein that contains a forkhead-associated (FHA) domain and two BRCA1 carboxy-terminal (BRCT) domains, as a binding partner for the MRE11 complex. We show that, in response to ionizing radiation, MDC1 is hyperphosphorylated in an ATM-dependent manner, and rapidly relocalizes to nuclear foci that also contain the MRE11 complex, phosphorylated histone H2AX and 53BP1. Downregulation of MDC1 expression by small interfering RNA yields a radio-resistant DNA synthesis (RDS) phenotype and prevents ionizing radiation-induced focus formation by the MRE11 complex. However, downregulation of MDC1 does not abolish the ionizing radiation-induced phosphorylation of NBS1, CHK2 and SMC1, or the degradation of CDC25A. Furthermore, we show that overexpression of the MDC1 FHA domain interferes with focus formation by MDC1 itself and by the MRE11 complex, and induces an RDS phenotype. These findings reveal that MDC1-mediated focus formation by the MRE11 complex at sites of DNA damage is crucial for the efficient activation of the intra-S-phase checkpoint.

• MeSH
• buněčné linie MeSH
• checkpoint kinasa 2 MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• enzymy opravy DNA MeSH
• fosfatasy cdc25 MeSH
• fosforylace účinky záření   MeSH
• ionizující záření MeSH
• jaderné proteiny chemie   metabolismus   MeSH
• lidé MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• poškození DNA * MeSH
• protein-serin-threoninkinasy * MeSH
• proteinkinasy metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• S fáze MeSH
• terciární struktura proteinů MeSH
• trans-aktivátory chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH