Podcasts about mre11

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.27.546694v1?rss=1 Authors: Bedir, M., Outwin, E., Colnaghi, R., Bassett, L., Abramowicz, I., O'Driscoll, M. Abstract: We previously reported that non-homologous end-joining (NHEJ)-defective human LIG4-/- pre-B lymphocytes were unexpectedly sensitive to killing by the cyclic peptide Cyclosporin A (CsA), a common component of bone marrow transplantation conditioning and maintenance regimes. We also found that CsA induced DNA double strand breaks (DSBs) in LIG4 syndrome patient fibroblasts, specifically upon transit through S-phase. The molecular basis underlying these CsA impacts has not been described hitherto. We postulated that CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair, as CYPA is the primary physiological target interactor of CsA. CYPA is the founding member of the Cyclophilin family of peptidyl-prolyl cis-trans isomerases (PPIs). CsA inhibits the PPI activity of CYPA through occupation of the latters enzymatic active site. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression-interaction analysis, we describe novel impacts of CYPA loss and inhibition of its PPI activity on DNA repair. Prompted by findings from our CYPA-BioID proximity interactome, we validate CYPA interactions with different components of the DNA end resection machinery. Moreover, we characterise a novel and direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 (MRN) complex, providing evidence that the PPI function of CYPA actively influences DNA repair via direct protein-protein interaction at the level of DNA end resection. Consequently, we demonstrate that CYPA loss or inhibition impairs Homologous Recombination Repair (HRR) following DNA replication fork stalling. Additionally, we define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability herein. Leveraging the novel insights into CYPA biology we have uncovered; we explore examples of how CYPA PPI inhibition may be exploited to selectively kill cells from a variety of different cancers with a shared characteristic genomic instability profile. These findings propose a potential new disease application or repurposing strategy for the non-immunosuppressive CsA analogue class of Cyclophilin inhibitors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.09.519750v1?rss=1 Authors: Cho, M.-G., Kumar, R. J., Lin, C.-C., Boyer, J. A., Shahir, J. A., Fagan-Solis, K., Simpson, D. A., Fan, C., Foster, C. E., Goddard, A. M., Wang, Q., Wang, Y., Ho, A. Y., Liu, P., perou, c. J., Zhang, Q., McGinty, R. K., Purvis, J. E., Gupta, G. P. Abstract: Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS/STING-mediated innate immune signaling and tumor suppression1-3. However, the mechanism for cGAS activation by endogenous DNA damage remains enigmatic, particularly given the constitutive inhibition of cGAS by high-affinity histone acidic patch (AP) binding4-10. Here we report an in vivo CRISPR screen that identified the DNA double strand break sensor Mre11 as a suppressor of mammary tumorigenesis induced by Myc overexpression and p53 deficiency. Mre11 antagonizes Myc-induced proliferation through cGAS/STING activation. Direct binding of the Mre11-Rad50-Nbn (MRN) complex to nucleosomes displaces cGAS from AP sequestration, which is required for DNA damage-induced cGAS mobilization and activation by cytosolic DNA. Mre11 is thereby essential for cGAS activation in response to oncogenic stress, cytosolic DNA transfection, and ionizing radiation. Furthermore, we show Mre11-dependent cGAS activation suppresses Myc-induced proliferation through ZBP1/RIPK3/MLKL-mediated necroptosis. In human triple-negative breast cancer, ZBP1 downregulation correlates with increased genome instability, decreased immune infiltration, and poor patient prognosis. These findings establish Mre11 as a critical link between DNA damage and cGAS activation that regulates tumorigenesis through ZBP1-dependent necroptosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

The integrity of the genome displays a central role for all living organisms. Double strand breaks (DSBs) are probably the most cytotoxic and hazardous type of DNA lesion and are linked to cancerogenic chromosome aberrations in humans. To maintain genome stability, cells use various repair mechanisms, including homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways. The Mre11:Rad50 (MR) complex plays a crucial role in DSB repair processes including DSB sensing and processing but also tethering of DNA ends. The complex consists of the evolutionarily conserved core of two Rad50 ATPases from which a long coiled-coil region protrudes and a dimer of the Mre11 nuclease. Even though various enzymatic and also structural functions of MR(N) could be determined, so far the molecular interplay of Rad50´s ATPase together with DNA binding and processing by Mre11 is rather unclear. The crystal structure of the bacterial MR complex in its nucleotide free state revealed an elongated conformation with accessible Mre11 nuclease sites in the center and a Rad50 monomer on each outer tip, thus suggesting conformational changes upon ATP and/or DNA binding. However, so far high resolution structures of MR in its ATP and/or DNA bound state are lacking. The aim of this work was to understand the ATP-dependent engagement-disengagement cycle of Rad50´s nucleotide binding domains (NBDs) and thereby the ATP-controlled interaction between Mre11 and Rad50. For this purpose high resolution crystal structures of the bacterial Thermotoga maritima (Tm) MR complex with engaged Rad50 NBDs were determined. Small angle x-ray scattering proved the conformation of the nucleotide bound complex in solution. DNA affinity was also analyzed to investigate MR´s DNA binding mechanism. ATP binding to TmRad50 induces a large structural change and surprisingly, the NBD dimer binds directly in the Mre11 DNA binding cleft, thereby blocking Mre11’s dsDNA binding sites. DNA binding studies show that MR does not entrap DNA in a ring-like structure and that within the complex Rad50 likely forms a dsDNA binding site in response to ATP, while the Mre11 nuclease module retains ssDNA binding ability. Finally, a possible mechanism for ATP dependent DNA tethering and DSB processing by MR is proposed.

Thu, 21 Jul 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13400/ https://edoc.ub.uni-muenchen.de/13400/1/Schiller_Christian_Bernd.pdf Schiller, Christian Bernd ddc:540, ddc:500, Fakultät für Chem

DNA damage poses a considerable threat to genomic integrity and cell survival. One of the most harmful forms of DNA damage are double-strand breaks that arise spontaneously during regular DNA processing like replication or meiosis. In addition, they can also be induced by a variety of DNA damaging agents like UV light, cell toxins or anti-cancer drugs. Failure of the rapid repair of these breaks can lead to chromosomal rearrangements and ultimately tumorigenesis in humans. In response to these genomic threats, a highly developed DNA repair network of protein factors has evolved, where the Mre11/Rad50/Nbs1 (MRN) complex is sought to play a key role in sensing, processing and repair of DNA double-strand breaks. Orthologs of Mre11 and Rad50, but not Nbs1, are found in all taxonomic kingdoms of life, suggesting that Mre11 and Rad50 form the core of this complex. In this work structural studies were performed to decipher the overall architecture and the interaction of SbcC and SbcD, the bacterial orthologs of Rad50 and Mre11. Using X-ray crystallographic and small angle X-ray scattering techniques the crystal as well as the in solution structures of the Thermotoga maritima SbcC ATPase domain in complex with full-length SbcD were solved. The crystal and in solution structure match well fortifying the calculated models that reveal an open, elongated complex with dimensions of approximately 210 Å * 75 Å * 65 Å. The heterotetrameric protein assembly consists of two SbcD molecules that homodimerize at domains I to form the central portion of the complex. Located at the outer areas of this homodimer domains II are arranged close to lobe II of SbcC building a small protein-protein interface. The C-terminal domains III of SbcD are connected to domains II via a flexible linker and associate through hydrophobic interactions with the coiled-coils of SbcC. These arrangements in combination with earlier findings lead to a model where upon ATP-binding the complex performs a conformational switch resulting in a ring-shaped structure. This conformation would bear a central cavity to harbor DNA strands that can be processed by the inwards oriented nuclease active sites of SbcD.

Der Erhalt der genomischen Integrität ist für das Überleben von Organismen notwendig, jedoch können verschiedene DNA-Läsionen das genetische Material gefährden. DNA-Doppelstrangbrüche (DSB) stellen dabei eine besonders toxische DNA-Läsion dar, und schon ein einzelner DSB kann bei ausbleibender oder fehlerhafter Reparatur zum Absterben der Zelle führen. In höheren Eukaryonten gibt es zwei Mechanismen für die Reparatur eines DSB: nicht-homologe Endverknüpfung und homologe Rekombination. Bei der homologen Rekombination spielt der Rekombinationsfaktor Rad52 eine zentrale Rolle und wurde zu Beginn dieser Arbeit als ein Substrat für eine posttranslationale Modifikation mit SUMO identifiziert. Daraufhin wurde die Regulation von Rad52 durch die Modifikation mit SUMO untersucht. So konnte im Rahmen dieser Arbeit gezeigt werden, dass die SUMOylierung von Rad52 in Saccharomyces cerevisiae hauptsächlich an zwei nicht konservierten Lysinresten außerhalb der hoch konservierten Rad52-Domäne erfolgt und eng an Rekombinations- und DNA-Reparaturereignisse gekoppelt ist. So wird die Rad52-SUMOylierung durch enzymatische DSB während der Meiose und durch chemisch induzierte DSB in mitotischen Zellen ausgelöst. Hierfür ist der MRX-Komplex (bestehend aus Mre11, Rad50 und Xrs2) notwendig, der vor Rad52 im Rekombinationsprozess aktiv ist. Des Weiteren zeigt die vorliegende Arbeit, dass Zellen mit einer Rad52-Mutante, die nicht mehr mit SUMO modifiziert werden kann, keine auffälligen Wachstumsdefekte aufweisen, beispielsweise weder in Gegenwart DNA-schädigender Agenzien noch in der Meiose. Allerdings hat die SUMOylierung einen pro-rekombinatorischen Einfluss auf Rad52. Denn zum einen können Zellen, in denen zwei der Helikasen Rrm3, Sgs1 oder Srs2 deletiert sind, in Gegenwart von SUMOylierungsdefizientem Rad52 wachsen, da vermutlich keine toxischen Rekombinationsintermediate mehr entstehen wie in Gegenwart von Wildtyp Rad52. Zum anderen weisen Zellen mit SUMOylierungsdefizientem Rad52 Defekte bei speziellen Rekombinationsreaktionen auf. Die SUMOylierung schützt Rad52 zudem vor dem Abbau durch das Proteasom und ist besonders für die Rad52-Moleküle relevant, die am Rekombinationsgeschehen beteiligt sind. Diese Arbeit zeigt somit, dass die SUMOylierung von Rad52 die Aktivität des Rekombinationsfaktors dadurch reguliert, dass die im Rekombinationsprozess involvierten Rad52-Moleküle vor einem vorzeitigen Abbau geschützt werden.

Modern light microscopical techniques were employed to follow dynamical nuclear processes during the cell cycle and during DNA-repair. Laser-UVA-microirradiation The protein Rad51 is essential for the repair of double-strand breaks (DSBs) via the conservative homologous recombination repair pathway. To test the hypothesis that Rad51 localizes to damaged sites during DSB repair, a laser-UVA-microirradiation system was established. With this system spots with sizes around 1 µm in nuclei of living cells can be irradiated with UVA-light. After sensitization of cells by incorporation of BrdU into nuclear DNA and staining with the live cell dye Hoechst 33258, the system can be used to introduce double-strand breaks and single-strand breaks in the irradiated spots. The response of Rad51 to microirradiation By use of laser-UVA microirradiation the localization of Rad51 at damaged sites containing DNA double-strand breaks could be demonstrated. The accumulation of Rad51 at microirradiated sites was followed in cells fixed at increasing times after microirradiation. First Rad51 accumulations were visible 5 - 10 minutes after irradiation, and the number of cells with Rad51 accumulations increased until a plateau was reached 20 - 30 minutes after irradiation. In contrast, the majority of irradiated cells had accumulations of Mre11 protein already 5 - 10 minutes after irradiation. This is consistent with reports that nuclear Mre11 foci appeared early in the response to ionizing radiation, but absolute response times were faster after microirradiation than after ionizing radiation. Large-scale nuclear patterns were microirradiated, and Rad51 accumulations that reflected the shape of the irradiated patterns were found up to eight hours after irradiation. This conservation of the pattern of Rad51 accumulations, which reflect sites containing the damaged DNA, indicated that the chromatin in the irradiated cells performs no large scale reordering in response to DNA damage. The dynamics of chromosomes and chromosome territories In 1909 Theodor Boveri forwarded the hypothesis that arrangements of chromosome territories (CTs) are stably maintained during interphase, but subject to changes during mitosis. In the last decade several groups reported evidence for the stability of CT arrangements, but considerable movements of chromosomal subregions were also observed. The data concerning the maintenance or reordering of CTs during mitosis have been contradictory. Live cell imaging To follow the movements of chromosomes and CTs, a novel experimental approach was taken. Cells expressing a fusion protein of the core histone H2B with GFP (H2B-GFP) stably incorporate H2B-GFP into nucleosomes. In these cells chromatin regions were selectively marked by laser-photobleaching and followed by live cell microscopy. To this end, a live cell imaging system was established at a confocal laser-scanning microscope, which allows the observation of living cells for several days. Chromatin movements visualized by photobleached H2B-GFP To track possible movements in interphase cell nuclei, stripe patterns were bleached into nuclei at several stages of interphase. These patterns were retained for up to two hours, until they became invisible due to the replacement of bleached H2B-GFP by unbleached H2B-GFP, supporting the hypothesis that CT order is stably maintained during interphase. Nuclei, in which all chromatin except for a contiguous zone at one nuclear pole was bleached, were followed through mitosis. At prophase a number of unbleached chromosomal segments became visible. The segments showed a variable degree of clustering in metaphase. When daughter nuclei were formed, the segments locally decondensed into patches of unbleached chromatin. In all daughter cells the patches were separated by bleached chromatin, and clustered to a variable extent. These observations support the hypothesis that changes of chromosome neighborhoods occur during mitosis and that CT neighborhoods can profoundly vary from one cell cycle to the next.