Podcasts about nhej

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.02.547417v1?rss=1 Authors: Kato-Inui, T., Ono, T., Miyaoka, Y. Abstract: As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 exhibited the improved HDR/NHEJ ratio better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing. 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/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/2023.06.27.546688v1?rss=1 Authors: Sutcu, H., Rassinoux, P., Donnio, L.-M., Neuillet, D., Vianna, F., Gabillot, O., MARI, P.-O., Baldeyron, C., Giglia-Mari, G. Abstract: DNA integrity is incessantly confronted to endogenous and exogenous agents inducing DNA lesions, which are harmful for cellular homeostasis. Luckily all organisms are equipped with a network of DNA damage response (DDR) mechanisms that will repair DNA lesions and restore the proper cellular activities. Despite DNA repair mechanisms have been revealed in vitro and in replicating cells, still little is known on how DNA lesions are repaired and consequently how cellular homeostasis is maintained in post-mitotic cells. Muscle fibers are highly specialised post-mitotic cells organized in syncytia and, they are vulnerable to age-related degeneration and atrophy following radiotherapy treatment. We have here studied in detail the DNA repair capacity of muscle fibers nuclei and compared it with the one measured in proliferative myoblasts. We focused on the DNA repair mechanisms that correct ionizing radiation (IR)-induced lesions, namely the base excision repair (BER), the non-homologous end joining (NHEJ) and the homologous recombination (HR). We found that in the most differentiated myogenic cells, myotubes, all of these DNA repair mechanisms present weakened kinetics of recruitment of DNA repair proteins to IR-damaged DNA. For BER and HR, this decline can be linked to reduced steady state levels of key proteins involved in these processes, probably since nuclei within muscle fibers no longer replicate their DNA. 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/2023.06.22.546088v1?rss=1 Authors: Mikhova, M., Heyza, J. R., Meek, K., Schmidt, J. C. Abstract: Non-homologous end joining (NHEJ) is the predominant path-way that repairs DNA double-stranded breaks (DSBs) in vertebrates. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed approximately 115[A] apart in one of two long-range synaptic complexes. The first long-range complex can be formed with only the KU70/80 heterodimer and DNA-PKcs while the second long-range complex also includes XRCC4, XLF, and Ligase 4. Both long-range complexes inefficiently progress to short-range synaptic complexes that juxtapose DNA ends to facilitate ligation. Here we perform singlemolecule analyses of the recruitment of Halo-tagged NHEJ factors to DSBs. Our results provide direct evidence for stepwise maturation of NHEJ complex and precisely define kinetics of core NHEJ factor binding to DSBs in living cells. 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/2023.03.17.533134v1?rss=1 Authors: Gelot, C., Kovacs, M. T., Miron, S., Mylne, E., Ghouil, R., Popova, T., Dingli, F., Loew, D., Guirouilh-Barbat, J., Del Nery, E., Zinn-Justin, S., Ceccaldi, R. Abstract: DNA double strand breaks (DSBs) are deleterious lesions that challenge genome integrity. To mitigate this threat, human cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle. In interphase, DSBs are mainly repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). However, these pathways are completely inhibited in mitosis, leaving the fate of mitotic DSBs unknown. Here we show that DNA polymerase theta (Pol{theta}) repairs mitotic DSBs and thereby maintains genome integrity. In contrast to other DSB repair factors, Pol{theta} function is activated in mitosis upon phosphorylation by the Polo-like kinase 1 (PLK1). Phosphorylated Pol{theta} is recruited to mitotic DSBs, where it mediates joining of broken DNA ends, while halting mitotic progression. The lack of Pol{theta} leads to a shortening of mitotic duration and defective repair of mitotic DSBs, resulting in a loss of genome integrity. In addition, we identify mitotic Pol{theta} repair as the underlying cause of the synthetic lethality between Pol{theta} and HR. Our findings reveal the critical importance of mitotic DSB repair for maintaining genome stability. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Tina and Hillary cover George W. Lee and Steve Lough. Tina's Story George Wesley Lee was a prominent civil rights leader in Humphreys County Mississippi. BUT when he continued to push for equal voting rights for blacks, he was murdered. Hillary's Story Steve Lough worked for years with the Ringling Brothers circus. BUT when he saw bozos like Trump running for office, he decided that a real clown should join the clowns in Congress. Sources Tina's Story AFL-CIO Black History Month Profiles: Rev. George W. Lee (https://aflcio.org/2020/2/13/black-history-month-profiles-rev-george-w-lee)--by Kenneth Quinnell Black Past GEORGE WASHINGTON LEE (1903-1955) (https://www.blackpast.org/african-american-history/people-african-american-history/george-washington-lee-1903-1955/)--by Samuel Momodu Mississippi Encyclopedia George Wesley Lee (Minister and Activist) (https://mississippiencyclopedia.org/entries/george-wesley-lee-minister-activist/) Southern Poverty Law Center REV. GEORGE LEE (https://www.splcenter.org/rev-george-lee) United States Department of Justice George Lee--Notice to Close File (Jul 12 2011) (https://www.justice.gov/crt/case-document/george-lee) Zinn Education Project May 7, 1955: Murder of Rev. George W. Lee (https://www.zinnedproject.org/news/tdih/rev-george-lee/) Photos George W. Lee (https://upload.wikimedia.org/wikipedia/en/a/aa/George%2BW.%2BLee.jpg)--from beejae.com (fair use) via Wikipedia Segregated Movie Theater in Belzoni (https://upload.wikimedia.org/wikipedia/commons/thumb/f/ff/Segregated_movie_theater.jpg/1920px-Segregated_movie_theater.jpg)--by Marion Post Wolcott (public domain) via Wikipedia Katherine Blair Saw Gunman's Car (https://s36500.pcdn.co/wp-content/uploads/2016/05/RevGeorgeLee_jet.jpg)--screenshot from 1955 Jet Magazine via Zinn Education Project Hillary's Story AP News Circus clown who ran for Congress dies in South Carolina (https://apnews.com/article/96c0c48bfcd24a88b43c64839aae13bd) Ballotpedia South Carolina's 5th Congressional District (https://ballotpedia.org/South_Carolina%27s_5th_Congressional_District) CBS News Why an Ivy League-educated former clown is running for Congress (https://www.cbsnews.com/news/why-an-ivy-league-educated-former-clown-is-running-for-congress/)--by Kathryn Watson CNN A clown is running for Congress in South Carolina (https://www.cnn.com/2018/03/27/politics/congress-south-carolina-clown-candidate/index.html)--by Veronica Stracqualursi Daily News Professional clown Steve Lough is dead, months after losing his bid for the U.S. House of Representatives (https://www.nydailynews.com/news/politics/ny-news-clown-south-carolina-dead-20190303-story.html) Facebook Steve Lough for Congress (https://www.facebook.com/ClownForCongress/) IBTimes UK Former clown Steve Lough running for Congress (https://www.youtube.com/watch?v=lTFLg8B8CAY) Kornegay Funeral Home Obituary Stephen Harrison "Steve" Lough of Camden, South Carolina (http://www.kornegayfuneralhomes.com/obituary/stephen-harrison-steve-lough) WIS News 10 So, there is a clown running for Congress in South Carolina (https://www.wistv.com/story/37809716/so-there-is-a-clown-running-for-congress-in-south-carolina/)--by Tanita Gaither Photos Steve Lough (https://www.wistv.com/story/37809716/so-there-is-a-clown-running-for-congress-in-south-carolina/)--screenshot via WIS News 10 Steve Lough in Clown Costume (https://www.gannett-cdn.com/authoring/2018/03/26/NHEJ/ghows-NC-cdda3d43-8dcf-44b8-8e71-36ae8cbb4dfa-74becdce.jpeg?width=660&height=495&fit=crop&format=pjpg&auto=webp)--from Notes on a Cowardly Lion via GoUpstate Lough Campaign Website (https://www.counton2.com/wp-content/uploads/sites/7/2018/03/steve20lough20congress_1522183598151.JPG_38541363_ver1.0.jpg)--screenshot of homepage via News 2 NBC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.06.527258v1?rss=1 Authors: He, J., Guo, Y., Deng, R., Li, L., Huang, C., Chen, R., Wang, Y., Huang, J., Cheng, J., Chen, G.-Q., Zheng, J., Zhao, X., Yu, J. Abstract: Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and cell survival. Nuclear PTEN plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses non-homologous end joining (NHEJ) repair by directly dephosphorylating 53BP1. During DNA damage responses (DDR), p14ARF was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DNA-break sites. This was because that SUMO1 conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of BRCA1, which has been located to the core of 53BP1 foci on the chromatin during S/G2 stage. Further, these chromatin-loaded PTEN directly and specifically dephosphorylated pT543 of 53BP1, resulting in the dissociation of 53BP1-complex, which facilitated DNA end resection and ongoing HR repair. The SUMOylation-deficient PTENK254R mice also showed decreased DNA damage repair in vivo. Blocking PTEN SUMOylation pathway with either an SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides the new mechanistic understanding of PTEN in HR repair and clinical intervention of chemo-resistant tumors. 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/2022.11.30.518500v1?rss=1 Authors: Braun, H., Xu, Z., Chang, F., Viceconte, N., Rane, G., Levin, M., Lototska, L., Roth, F., Hillairet, A., Fradera-Sola, A., Khanchandani, V., Dreesen, O., Yang, Y., Shi, Y., Li, F., Butter, F., Kappei, D. Abstract: Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing non-homologous end joining (NHEJ) through its subunit TRF2. We here describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity and reveal the base-specific sequence recognition by co-crystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

BUFFALO, NY- June 16, 2022 – A new editorial paper was published in Aging (Aging-US) Volume 14, Issue 11, entitled, “WRNing for the right DNA repair pathway choice.” Premature aging diseases, also called ‘progeroid syndrome', display signs and features of normal aging in early life, ultimately leading to premature death. Although progeroid syndromes do not perfectly mimic chronological aging, they can be excellent model systems to study characteristics of normal aging. Werner syndrome (WS) is one of the rare autosomal recessive progeroid syndromes, characterized by accelerated aging. WRN is suggested to play a central role in maintaining genome stability and rapidly recruits to the DNA damage sites to take part in DNA repair, including base excision DNA repair (BER), classical/alternative non-homologous end joining (NHEJ), homologous recombination (HR), and replication re-start after DNA damage. WRN makes critical DNA-repair pathway choices between classical and alternative NHEJs. In addition to its key role in NHEJ, WRN has been suggested to also participate in HR. However, how it regulates the pathway choice between NHEJ and HR was still unclear. Full press release - https://aging-us.net/2022/06/16/aging-us-wrning-for-the-right-dna-repair-pathway-choice/ DOI: https://doi.org/10.18632/aging.204120 Corresponding Author: Vilhelm A. Bohr - vbohr@nih.gov Keywords: DNA repair, RecQ helicase, helicase, DNA double strand repair pathways Sign up for free Altmetric alerts about this article: https://aging.altmetric.com/details/email_updates?id=10.18632%2Faging.204120 About Aging-US: Launched in 2009, Aging (Aging-US) publishes papers of general interest and biological significance in all fields of aging research and age-related diseases, including cancer—and now, with a special focus on COVID-19 vulnerability as an age-dependent syndrome. Topics in Aging go beyond traditional gerontology, including, but not limited to, cellular and molecular biology, human age-related diseases, pathology in model organisms, signal transduction pathways (e.g., p53, sirtuins, and PI-3K/AKT/mTOR, among others), and approaches to modulating these signaling pathways. Follow Aging on social media: SoundCloud – https://soundcloud.com/Aging-Us Facebook – https://www.facebook.com/AgingUS/ Twitter – https://twitter.com/AgingJrnl Instagram – https://www.instagram.com/agingjrnl/ YouTube – https://www.youtube.com/agingus​ LinkedIn – https://www.linkedin.com/company/aging/ Pinterest – https://www.pinterest.com/AgingUS/ For media inquiries, please contact media@impactjournals.com.

As the year comes to an end, we would like to thank everyone who listened to the FYI — For Your Innovation podcast. 2021 was another up and down year, as the world continues to battle the COVID-19 pandemic. In this final episode of 2021, we compiled some of our most interesting podcast episodes for you. Please enjoy this summary and tune back in when we return in 2022 with a new and improved version of FYI. Check out the FYI – For Your Innovation Podcast ‘Best of 2021'. Because investing in innovation starts with understanding it. #FYIpodcast Key Points From This Episode: Factors that caused CRISPR to dominate the gene editing space. David explains the difference between DNA and RNA, using a football analogy. The goal that David centered his PhD around. Variety that exists amongst RND molecules. Benefits of targeting RNA in diseased cells, rather than DNA. Why RNA lagged behind DNA in terms of technological developments. A theory about the role played by RNA in the past. Methods of RNA editing. Comparing homology directed repair (HDR) and non homologous end joining (NHEJ). David provides a breakdown of the various different guides and enzymes. Three kinds of vector systems, and how they work. Examples of the types of RNAs that exist. What David is excited about in the future of the RNA technology field.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.381160v1?rss=1 Authors: Liu, L., Li, K., Zou, L., Hou, H., Hu, Q., Liu, S., Wang, S., Wang, Y., Li, J., Song, C., Chen, J., Li, C., Du, H., Li, J.-L., Chen, F., Xu, Z., Sun, W., Sun, Q., Xiong, W. Abstract: Frameshift mutation caused by small insertions/deletions (indels) often generate truncated and non-functional proteins, which underlies 22% inherited Mendelian disorders in humans. However, there is no efficient in vivo gene therapy strategy available to date, especially in postmitotic systems. Here, we leveraged the non-homologous end joining (NHEJ) mediated non-random editing profiles to compensate the frameshift mutation in a USH1F mouse model - av3j. After treatment by the selected gRNA, about 50% editing products showed reading-frame restoration, and more than 70% targeted hair cells recovered mechanotransduction. In vivo treatment ameliorated the hearing and balance symptoms in homozygous mutant mice. Furthermore, a scale-up analysis of 114 gRNAs targeting 40 frameshift deafness mutations reveals that 65% loci have at least one gRNA with predicted therapeutic potential. Together, our study demonstrates that the NHEJ-mediated frame restoration is a simple and highly efficient therapeutic strategy for small-indel induced frameshift mutations. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.382283v1?rss=1 Authors: Kurgan, G., Turk, R., Li, H., Rettig, G. R., Jacobi, A. M., Tso, L., Mertens, M., Noten, R., Florus, K., Behlke, M. A., Wang, Y., McNeill, M. S. Abstract: CRISPR systems enable targeted genome editing in a wide variety of organisms by introducing single- or double-strand DNA breaks, which are repaired using endogenous molecular pathways. Characterization of on- and off-target editing events from CRISPR proteins can be evaluated using targeted genome resequencing. We characterized DNA repair footprints that result from non-homologous end joining (NHEJ) after double stranded breaks (DSBs) were introduced by Cas9 or Cas12a for >500 paired treatment/control experiments. We found that building our understanding into a novel analysis tool (CRISPAltRations) improved results quality. We validated our software using simulated rhAmpSeq amplicon sequencing data (11 gRNAs and 603 on- and off-target locations) and demonstrate that CRISPAltRations outperforms other publicly available software tools in accurately annotating CRISPR-associated indels and homology directed repair (HDR) events. We enable non-bioinformaticians to use CRISPAltRations by developing a web-accessible, cloud-hosted deployment, which allows rapid batch processing of samples in a graphical user-interface (GUI) and complies with HIPAA security standards. By ensuring that our software is thoroughly tested, version controlled, and supported with a UI we enable resequencing analysis of CRISPR genome editing experiments to researchers no matter their skill in bioinformatics. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.27.354092v1?rss=1 Authors: Sowunmi, K., Soyebo, T. A., Okosesi, E. A., Adesiyan, A. L., Oladimeji, K. A., Ajibola, O. A., Ogunlana, Y. O., Agboola, O. W., Kaur, G., Atoromola, H., Oladipupo, T. A. Abstract: The development of precise DNA editing nucleases that induce double-strand breaks (DSBs) - including zinc finger nucleases, TALENs, and CRISPR/Cas systems - has revolutionized gene editing and genome engineering. Endogenous DNA DSB repair mechanisms are often leveraged to enhance editing efficiency and precision. While the non-homologous end joining (NHEJ) and homologous recombination (HR) DNA DSB repair pathways have already been the topic of an excellent deal of investigation, an alternate pathway, microhomology-mediated end joining (MMEJ), remains relatively unexplored. However, the MMEJ pathway's ability to supply reproducible and efficient deletions within the course of repair makes it a perfect pathway to be used in gene knockouts. (Microhomology Evoked Deletion Judication EluciDation) may be a random forest machine learning-based method for predicting the extent to which the location of a targeted DNA DSB are going to be repaired using the MMEJ repair pathway. On an independent test set of 24 HeLa cell DSB sites, MEDJED achieved a Pearson coefficient of correlation (PCC) of 81.36%, Mean Absolute Error (MAE) of 10.96%, and Root Mean Square Error (RMSE) of13.09%. This performance demonstrates MEDJED's value as a tool for researchers who wish to leverage MMEJ to supply efficient and precise gene knock outs. Copy rights belong to original authors. Visit the link for more info

And we're back with Part 2 of 2 with Owen Dunkley. This time we've got an alliterative bonus episode covering COVID, CRISPR and COVID! Here are some of the subtopics we hit on within each: CANCER: Mutations, somatic vs. germ-line cells, radiation therapy, chemotherapy, personalized medicine and the future of drug therapy. CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats, the CAS-9 complex, genomes, viruses and bacteria, plasmids, gene editing, NHEJ and HDR, prime editing. COVID: SARS-CoV-2 vs. SARS-CoV-1: the original SARS, positive single-stranded RNA viruses, infectious diseases, monitoring and the job of global health organizations We hope you enjoy Owen Dunkley in his appearance on this bonus episode, as he shares more of his invaluable knowledge on biological processes, diseases and viruses! --- Send in a voice message: https://anchor.fm/abstractcast/message

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.24.261875v1?rss=1 Authors: Gavande, N. S., VanderVere-Carozza, P. S., Pawelczak, K. S., Vernon, T. L., Hanakahi, L. A., Summerlin, M., Dynlacht, J. R., Farmer, A. H., Sears, C. R., Al Nasrallah, N., Garrett, J., Turchi, J. J. Abstract: DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku-DNA interaction. We have developed a series of highly potent and specific Ku-DNA binding inhibitors (Ku-DBi's) that block the Ku-DNA interaction and inhibit DNA-PK kinase activity. Ku-DBi's directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the activity of IR and radiomimetics. Analysis of Ku-null cells demonstrates that Ku-DBi's cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBi's. The utility of Ku-DBi's was also demonstrated in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBi's, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.29.227033v1?rss=1 Authors: Carney, S. M., Moreno, A. T., Piatt, S. C., Cisneros-Aguirre, M., Lopezcolorado, F. W., Stark, J. M., Loparo, J. J. Abstract: Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation. Copy rights belong to original authors. Visit the link for more info

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.

Il processamento degli antigeni Le cellule APC Presentazione di proteine citosoliche Il trasportatore

Rad5 is a decisive protein in S. cerevisiae due to its role in the Post-replication repair (PRR) pathway, in which Rad5 is necessary for at least one error-free and one error-prone repair subpathway. In addition, Rad5 plays a role in other repair pathways; for instance, Rad5 regulates the balance between the double strand break (DSB) repair pathways, favoring the Rad52-dependent Homologous Recombination (HR) over the yKu70-dependent Non-Homologous-End Joining (NHEJ). Furthermore, since UV-induced damages are substrates for Rad5 but also for Base Excision Repair (BER) proteins, Rad5 is possibly involved directly or indirectly in the BER pathway. To get a deeper insight into the interaction of Rad5 with HR, NHEJ and BER proteins, survival curves, plasmid assays, and mutagenicity experiments were carried out in this work. In addition, a new software tool has been developed for the quantification of DSB. This software, called Geltool, allows the quantification of DSB in haploid cells from PFGE gels, even if the number of DSB is small. This represents a decisive advantage in comparison with previous programs. The sensitivity of Geltool has permitted the quantification of DSB repair during the stationary growth phase in haploid cells, detecting a repair of 46 %- 57 % of the gamma-induced DSB in HR proficient strains against 6 % - 16 % in HR deficient strains. Studies of the functional interactions of Rad5 with HR and NHEJ proteins revealed a synergistic effect between Rad5 and Rad52 proteins for the repair of DSB at chromosomal and plasmidial level. Differences in the repair of plasmids from the rad52 and the rad5 mutants revealed different end joining mechanisms for gap repair. Severe degradations found in plasmids from rad52 and rad52rad5 mutants could indicate an end protection function for Rad52 and also for Rad5, when Rad52 is absent. Moreover, the regulatory role of the Rad5 protein is confirmed, since the additional deletion of YKU70 suppresses the rad5 phenotype and forces the yku70rad5 mutant to repair by HR. The further study of the interplay of Rad5 with BER proteins shows that while BER only plays a minor role for the repair of gamma-induced damage, the rad5 phenotype is suppressed in the BER deficient apn1ntg1ntg2rad5 mutant. The same phenotype of suppression is also found for survival after UV irradiation. An enhanced mutagenicity of the apn1ntg1ntg2rad5 mutant indicates a possible repair through the REV3-dependent Translesion Synthesis Repair (TLS) pathway, suggesting that an error-prone tolerance of UV-induced damage can be very effective for survival.

The Ku protein from Saccharomyces cerevisiae (Yku) forms, like its human homologue hKu, a heterodimer comprised of a 70kD and an 80 kD subunit. In yeast and mammals, the Ku heterodimer is required for the repair of DNA double strand breaks (DSBs) via nonhomologous end-joining (NHEJ). Interestingly, Ku has been shown to bind to the native chromosome ends. It contributes to the maintenance of wild type telomere length and, moreover, has been implicated in the protection of the telomeres from end-to-end fusions. Telomere-bound Yku delocalizes from telomeric foci in response to DNA damage and accumulates at the sites of a DNA break. This thesis aimed to further characterize the Yku heterodimer and its function at DSBs and the native chromosome ends. In a genetic screen for mutations that - in combination with a yku deletion - lead to cell death, a novel mutation in the yeast telomerase subunit CDC13/EST4 has been identified earlier in the laboratory. Cdc13p binds to the single stranded DNA overhang at telomeres and is required to recruit the yeast telomerase to chromosome ends. The results presented here suggest that the mutant protein, Cdc13-4p, can still bind to the telomere and does interact with the telomerase subunit Est1p in vivo. A model is proposed in which the mutant Cdc13p is altered in its binding to a regulatory protein, thereby modulating telomerase access to the chromosome ends. The lethal effect in yku mutants is discussed to result from the loss of additional telomere sequences at the already very short telomeres of yku mutants. In order to fulfill its opposite functions at the ‘different’ DNA ends, Yku might depend on larger protein networks. Putative Yku interacting proteins have been identified in a two hybrid screen. One interactor, Sir4p, has previously been implicated in NHEJ. The Sir4p domain identified could be shown to interact with the Yku heterodimer via the Yku80p subunit. Experiments that allowed the separation of phenotypes caused by the loss of the Sir4 protein itself and phenotypes induced by a de-repression of silencing in sir4 mutants revealed no direct involvement of Sir4p in the repair of DSBs. In contrast to Cdc13p, Sir4p acts epistatic with Yku at the telomeres, indicating that the protein-protein interaction detected by two hybrid criteria might take place at telomeres. Besides defects in DNA repair and telomere protection, mice deficient for Ku have been reported to exhibit phenotypes indicative of premature aging. Loss of yku70 or overexpression of the Yku heterodimer effects life span in yeast. Experiments presented here rise the possibility that the premature aging is correlated with Ku’s function at the telomere.