The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a ‘head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damag

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

The CHD4 helicase is identified as a new component of the genome surveillance machinery in a proteomic screen for factors enriched on chromatin after ionizing radiation (see also related paper by Smeenk et al. in this issue)

The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a ‘head-to-tail’ dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage

The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks

Chromatin changes at sites of DNA double-strand breaks

Eukaryotic cells are constantly exposed to a multitude of genotoxic agents of either exogenous sources such as exposure to ultraviolet components of the sunlight, or of endogenous sources such as metabolically produced reactive oxygen species. The resulting lesions are a constant threat to the stability of the genome. DNA double-strand breaks (DSBs) are among the most cytotoxic types of DNA damage. They are induced by ionizing radiation or clastogens and can be generated by replication over a single-strand break. If left unrepaired they can lead to cell death or gross chromosomal aberrations, and promote the onset of diseases associated with genomic instability such as cancer. In mammalian cells, a global signalling network – called the DNA damage response (DDR) – has evolved that senses the DNA lesions and regulates appropriate responses such as DNA repair, transient or permanent cell cycle arrest, apoptosis or senescence as well as induction of certain transcriptional programs. In response to DSBs, the MRE11/RAD50/NBS1 (MRN) complex is involved in the recruitment and activation of the kinases ATM and DNA-PKcs that phosphorylate various target proteins. Among those is the histone variant H2AX, a natural component of chromatin that occurs in about 7-15% of all nucleosomes. The adaptor protein MDC1 directly binds to phosphorylated H2AX (γH2AX) and mediates recruitment and retention of other DDR factors in chromatin domains flanking the lesion. MDC1 is constitutively phosphorylated by casein kinase 2, which was shown to be required for the recruitment of the MRN complex to damaged chromatin via its subunit NBS1. NBS1 contains two phospho-specific interaction modules at its N-terminus, a FHA domain and a BRCT tandem domain. In this study, we show that both of these domains participate in the interaction with MDC1. Phospho-binding activities of both domains are essential for accumulation of the MRN complex in chromatin adjacent to DSBs in vivo. Surprisingly though, only mutation in the FHA domain, but not mutation in the BRCT domains, yields a G2/M checkpoint defect. Moreover, we did not detect increased radiosensitivity upon mutation of either the FHA or the BRCT tandem domain. Therefore, we concluded that MDC1-dependent retention of the MRN complex in the γH2AX-containing chromatin is not essential for a proper G2/M checkpoint activation, nor is it required for normal survival after ionizing radiation. Furthermore, we suggest that the FHA domain of NBS1 may have additional as yet unidentified binding partners that mediate G2/M checkpoint activation in response to DSBs. Therefore, we set out to search for such additional interaction partners. In fission yeast, NBS1 interacts with Ctp1 via the FHA domain, and this interaction is required for survival upon various DNA damaging agents. However, our preliminary data reveal that CtIP, the human orthologue of Ctp1, is rather not a direct binding partner of the FHA domain of human NBS1. Zellen sind ständig Substanzen ausgesetzt, die die DNA beschädigen. Diese können von der Umwelt (z.B. Sonnenstrahlung) oder durch den Organismus (z.B. freie Sauerstoffradikale) produziert werden. Die Gefahr besteht, dass die so entstanden DNA Schäden das Genom des Organismus verändern. Besonders Doppelstrangbrüche der DNA sind eine der grössten Bedrohungen. Sie entstehen durch die Einwirkung von ionisierender Strahlung oder auch durch die Replikation eines DNA Fragmentes mit einem Einzelstrangbruch. Falls Doppelstrangbrüche nicht repariert werden, können sie zum Zelltod oder zu Translokationen führen. Sie können auch zu Krankheiten wie Krebs führen. Menschliche Zellen besitzen spezialisierte Mechanismen zur Erkennung und Reparatur von DNA Schäden, sowie zur Aktivierung von Signalkaskaden, die das Fortschreiten des Zellzyklus hemmen, bestimmte Transkriptionsprogramme einleiten oder den programmierten Zelltod auslösen. Entstehen solche Doppelstrangbrüche, dann hilft der MRE11/RAD50/NBS1 (MRN) Komplex die beiden Kinasen ATM und DNA-PKcs zu aktivieren. Diese phosphorylieren dann unzählige Proteine, eines davon ist die Histone-Variante H2AX, die in 7-15% der Nukleosomen vorkommt. Das Adaptorprotein MDC1 bindet phosphoryliertes H2AX und führt zu weiterer Akkumulierung verschiedener Reparaturfaktoren im Chromatin in der Nähe des DNA Bruches. MDC1 wird von der Casein Kinase 2 phosphoryliert, was zur Rekrutierung vom MRN Komplex führt via seine Untereinheit NBS1. Am N-terminalen Ende von NBS1 sind zwei phosphospezifische Interaktionsdomänen lokalisiert, die FHA Domäne und die BRCT tandem Domäne. In dieser Studie zeigen wir, dass beide Domänen für die Interaktion zwischen MDC1 und dem MRN Komplex verantwortlich sind und somit auch beide für die Akkumulierung des MRN Komplexes im Chromatin gebraucht werden. Interessanterweise führt aber nur die Mutation einer konservierten Aminosäure in der FHA Domäne zu einem Defekt der Aktivierung des G2/M Zellzykluskontrollpunktes, nicht aber die Mutation in der BRCT Domäne von NBS1. Desweiteren konnten wir zeigen, dass Zellen mit den mutierten Domänen nicht radiosensitiver sind als Wildtypzellen. Daraus folgt, dass die Rekrutierung des MRN Komplexes nicht essenziell ist für die Aktivierung des G2/M Kontrollpunktes. Zusätzlich schlagen wir vor, dass NBS1 mit der FHA Domäne noch mit anderen Proteinen interagiert und dadurch den G2/M Kontrollpunkt aktiviert. Wir haben auch schon einige Kandidaten getestet. Es ist zum Beispiel bekannt, dass in Hefe die Interaktion zwischen Ctp1 und der FHA Domäne von NBS1 für die DNA Reparatur benötigt wird. Unsere ersten Ergebnisse zeigen jedoch, dass CtIP, das orthologe Protein in Menschenzellen, wahrscheinlich nicht mit der FHA Domäne von NBS1 interagiert