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
