MDC1: The art of keeping things in focus

The chromatin structure is important for recognition and repair of DNA damage. Many DNA damage response proteins accumulate in large chromatin domains flanking sites of DNA double-strand breaks. The assembly of these structures—usually termed DNA damage foci—is primarily regulated by MDC1, a large nuclear mediator/adaptor protein that is composed of several distinct structural and functional domains. Here, we are summarizing the latest discoveries about the mechanisms by which MDC1 mediates DNA damage foci formation, and we are reviewing the considerable efforts taken to understand the functional implication of these structure

A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma

BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease

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 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

Mechanism of DNA damage-induced MDC1 dimerization

Every cell needs to ensure the maintenance and faithful propagation of genetic information in order to prevent the development of life-threatening diseases such as cancer. However, cells are constantly exposed to various types of genotoxic agents e.g. free oxygen radicals or UV radiation. To counteract these threats, cells have developed specialized mechanisms to detect and repair DNA damage, and to initiate signaling cascades that delay or arrest cell cycle progression, induce certain transcriptional programs or trigger apoptosis. This complex signaling network is collectively denoted as the DNA damage response (DDR). Double strand breaks (DSBs) are one of the most hazardous forms of DNA damage because they can cause chromosomal instability or cell death if not repaired correctly. This type of DNA lesion can arise from the exposure to ionizing radiation or radiomimetic drugs, both frequently used in cancer treatment. DSBs lead to the activation of the ATM kinase and to the phosphorylation of the histone variant H2AX. Phosphorylated H2AX (γH2AX) is directly recognized by mediator of DNA damage checkpoint protein 1 (MDC1), a large mediator/adaptor protein that regulates the accumulation of DDR factors in chromatin regions flanking DSBs. MDC1 contains a forkhead associated (FHA) domain at its N-terminus. Even though it is known that FHA domains act as phosphopeptide recognition motifs, the function of the MDC1 FHA domain has remained enigmatic. In this study, we identified a novel phosphorylation-specific binding partner of the MDC1 FHA domain, namely MDC1 itself. We show that ATM phosphorylates MDC1 at the conserved N-terminal Thr4 residue in a DNA damage-dependent manner and that this induces the interaction with the FHA domain of other MDC1 molecules. Biochemical, biophysical and X-ray structural analysis revealed that the presence of a Thr4-phosphopeptide stabilizes the formation of a tight FHA dimer in a head-to-tail-oriented manner. We furthermore demonstrate that the isolated FHA domain is capable of localizing to sites of DNA damage in a phosphorylation-induced and dimerization-dependent manner. The elucidation of this mechanism contributes to our understanding of how MDC1 functions in the mammalian DDR and supports the notion that dimerization/oligomerization is a common theme of DDR mediator proteins. ZUSAMMENFASSUNG Der Erhalt und die getreue Weitergabe der genetischen Information ist wichtig für jede Zelle, um der Entwicklung von Krankheiten wie Krebs vorzubeugen. Zellen sind jedoch ständig verschiedenen Substanzen ausgesetzt, welche die DNA beschädigen (z.B. freie Sauerstoffradikale und UV-Strahlung). Um diese Gefahren abzuwenden, besitzen Zellen spezialisierte Mechanismen zur Erkennung und Reparatur von DNA Schäden sowie zur Aktivierung von Signalkaskaden, die das Fortschreiten des Zellzyklus hemmen, bestimmte Transkriptionsprogramme einleiten oder programmierten Zelltod (Apoptose) auslösen. Doppelstrangbrüche (DSBs) stellen dabei eine der gefährlichsten Formen von DNA Schäden dar, da sie bei nicht ausreichender Reparatur chromosomale Instabilität oder Zelltod zu verursachen vermögen. DSBs können durch die Einwirkung ionisierender Strahlung oder ähnlich wirkender Zytostatika, die beide häufig in der Krebstherapie ihre Anwendung finden, entstehen. DSBs führen zur Aktivierung der ATM-Kinase und Phosphorylierung der Histon-Variante H2AX. Phosphoryliertes H2AX wird direkt von MDC1, einem grossen Adapter-Protein, gebunden, das die Ansammlung von weiteren Proteinen in den Doppelstrangbruch umgebenden Chromatinregionen reguliert. MDC1 besitzt eine N-terminale FHA Domäne. Obwohl bekannt ist, dass FHA Domänen als Phosphopeptid-Erkennungsmotive fungieren, konnte die Funktion der MDC1 FHA Domäne bisher noch nicht entschlüsselt werden. In dieser Arbeit haben wir MDC1 selbst als einen neuen phosphospezifischen Interaktionspartner der MDC1 FHA Domäne identifiziert. Wir zeigen, dass MDC1 an der konservierten Aminosäure Thr4 infolge von DNA Schäden von der Kinase ATM phosphoryliert wird und dass dies für die Interaktion mit der FHA Domäne eines anderen MDC1 Moleküls verantwortlich ist. Biochemische, biophysikalische und Röntgenstruktur-Analysen legen offen, dass das T4-Phosphopeptid die Ausbildung eines FHA-Dimers in umgekehrter Orientierung stabilisiert. Des Weiteren kann die isolierte FHA Domäne an DNA geschädigte Regionen in Abhängigkeit vom Phosphorylierungs- und Dimerisierungsstatus binden. Die Aufklärung dieses Mechanismus trägt zu unserem Verständnis der Funktion von MDC1 in der Antwort auf DNA Schäden in Säugetierzellen bei und unterstützt die Auffassung von Dimerisierung (Oligomerisierung) als allgemeines Leitmotiv in Adapterproteinen.

Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest

The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function, and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein arginine methyltransferases; however, very little is known about which arginine residues become methylated on target substrates. Here we describe a proteomics methodology that combines single-step immunoenrichment of methylated peptides with high-resolution mass spectrometry to identify endogenous arginine mono-methylation (MMA) sites. We thereby identify 1027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared with the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers strong site-specific regulation of MMA sites during transcriptional arrest. Interestingly, several MMA sites are down-regulated after a few hours of transcriptional arrest. In contrast, the corresponding di-methylation or protein expression levels are not altered, confirming that MMA sites contain regulated functions on their own. Collectively, we present a site-specific MMA data set in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase