eSymposia | Genomic Stability and DNA Repair

Sep 21, 2020 ‐ Sep 23, 2020



Sessions

DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote Muravyova A., Alexander Smirnov, Veniamin Fishman, Anastasia Yunusova, Alexey Korablev, Irina Serova, Nariman Battulin Institute of Cytology and Genetics SB of RAS, Novosibirsk, Russia Mechanisms that ensure repair of double-strand DNA breaks (DSBs) are instrumental in the integration of foreign DNA into the genome of transgenic organisms. After pronuclear microinjection, exogenous DNA is usually found as a concatemer comprising multiple co-integrated transgene copies. Here, we investigated the contribution of various DSB repair pathways to the concatemer formation. We injected mouse zygotes with a pool of linear DNA molecules carrying unique barcodes at both ends and obtained 10 transgenic embryos with 1–300 transgene copies. Sequencing the barcodes allowed us to assign relative positions to the copies in concatemers and detect recombination events that occurred during integration. Cumulative analysis of approximately 1,000 integrated copies reveals that over 80% of them underwent recombination when their linear ends were processed by synthesis-dependent strand annealing (SDSA) or double-strand break repair (DSBR). We also observed evidence of double Holliday junction (dHJ) formation and crossing over during the concatemer formations. Sequencing indels at the junctions between copies shows that at least 10% of DNA molecules introduced into the zygotes are ligated by non-homologous end joining (NHEJ). We also injected the zygotes with circular DNA, Cas9 protein and gRNA and observed that Cas9 obstructs the concatemers formation. Our barcoding approach, verified with Pacific Biosciences Single Molecule Real-Time (SMRT) long-range sequencing, documents high activity of homologous recombination after DNA microinjection.

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Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance Anna Fortuny 1, Audrey Chansard 1, Pierre Caron 1, Odile Chevallier 1, Olivier Leroy 2, Olivier Renaud 2, Sophie E. Polo 1. 1-Epigenetics and Cell Fate Centre, UMR7216 CNRS, Université de Paris, F-75013 Paris, France. 2-Cell and tissue imaging facility, UMR3215 PICT-IBiSA, Institut Curie, F-75005 Paris, France. Heterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a system for targeting UV damage to pericentric heterochromatin in mammalian cells and for tracking the heterochromatin response to UV in real time. We uncover profound heterochromatin compaction changes during repair, orchestrated by the UV damage sensor DDB2, which stimulates linker histone displacement from chromatin. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We unveil a central role for the methyltransferase SETDB1 in the maintenance of heterochromatic histone marks after UV, SETDB1 coordinating histone methylation with new histone deposition in damaged heterochromatin, thus protecting cells from genome instability. Our data shed light on fundamental molecular mechanisms safeguarding higher-order chromatin integrity following DNA damage.

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At DNA damages, RNA polymerases-I fall off and are replaced by nucleosomes, concomitantly DNA repair switches from TC-NER to GG-NER

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

At DNA damages, RNA polymerases-I fall off and are replaced by nucleosomes, concomitantly DNA repair switches from TC-NER to GG-NER Audrey Paillé1, François Peyresaubes1, José Carlos Zeledon-Orellana1, Antonio Conconi1*, 1Université de Sherbrooke Nucleotide excision repair allows to remove a large variety of DNA damage, like pyrimidine dimers induced by UV radiations. These damages are extremely mutagen and obstruct the elongation of RNA polymerases. They are removed by the two sub-pathways of the nucleotide excision repair (NER): the global genome repair (GG-NER) that repairs most of the genome, and the transcription coupled repair (TC-NER) that only repairs the transcribed strand of active genes. How NER proceeds through non-nucleosomal chromatin and how open chromatin is reestablished after repair are widely unknown. In our lab, we analyzed NER in ribosomal genes (rDNA) which are present in multiple copies. The nucleolus is formed around clusters of rDNA that are transcribed by RNA polymerase I (RNAPI). The amount of rDNA varies among organisms, and there are ~150 genes in yeast. At any given time, only a portion of rDNA is transcribed and has no nucleosomes, whereas non-transcribed rDNA are folded in nucleosomes. In this study we want to understand the interplays between UV induced DNA damages, nucleotide excision repair and chromatin structure. Previously, we showed that RNAPI encountering DNA damage falls off the transcribed strand and is replaced by nucleosomes. Starting from this discovery, we want to know how the transcribed strand newly folded by nucleosomes is repaired. To answer this question, we employed a Primer Extension technique, based on DNA-polymerase extension of DNA primers to measure pyrimidine dimers and repair at the nucleotide level, in both strands of rRNA genes. In agreement with the current knowledge, the rDNA non-transcribed strand is repaired by GG-NER. Remarkably, we found that the transcribed-strand is not only repaired by TC-NER at the 5’-end but also by GG-NER towards the middle and at the 3’-end of the gene, mirroring the replacement of RNAPI by nucleosomes. These findings indicate that the nucleosomes folded on the transcribed strand induce a different NER sub-pathway rather than being a passive impediment. In perspective of this observation, we want to study more specifically the implication of nucleosomes and rDNA chromatin state after UV irradiation in the maintenance of genome stability.

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TRIP13-p31 is a negative regulator of the multifunctional REV7 protein

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

TRIP13-p31 is a negative regulator of the multifunctional REV7 protein Connor S. Clairmont*1, Prabha Sarangi*1, Lucas D. Galli1, Lisa A. Moreau1, Alan D. D’Andrea1,2 1. Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA 2. Center for DNA Damage and Repair, Dana-Farber Cancer Institute, Boston, MA, 02215, USA *These authors contributed equally to this work The REV7-Shieldin complex is an important component of the cellular machinery that controls DNA double strand break (DSB) repair. REV7-Shieldin is recruited to DSBs downstream of 53BP1 and counteracts DNA end resection, a prerequisite for repair by homologous recombination (HR). REV7 is a HORMA domain protein that mediates the critical interaction between SHLD3 and SHLD2. Similar to other HORMA domain proteins, REV7 binds to its partners through the closing of its “seatbelt” domain, resulting in extremely stable interactions. TRIP13 is a AAA+ ATPase that actively “opens” the seatbelt of REV7 and other HORMA domain proteins, thereby dissociating the involved complexes. As such, TRIP13 is an important regulator of HR activity - by dissociating the REV7-Shieldin complex, it promotes end resection and HR. Here we identify the small HORMA-like protein, p31comet as a critical adapter of the REV7-TRIP13 interaction. p31-deficient cells exhibit hyperactivation of the REV7-Shieldin complex, resulting in HR deficiency and hypersensitivity to poly-ADP ribose polymerase (PARP) inhibition. Conversely, overexpression of p31 leads to increased HR utilization, even in the otherwise HR-deficient context of BRCA1-knockout cells. Furthermore, p31 overexpression is frequently observed in cancers and correlates with prognosis and genomic mutation signatures, suggesting that it may be a determinant of mutagenesis and drug response in cancer.

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Acquired Resistance To BRAF Inhibitor Sensitizes Melanoma Cells To Chk1 Inhibition-Induced Replication Stress

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Acquired Resistance To BRAF Inhibitor Sensitizes Melanoma Cells To Chk1 Inhibition-Induced Replication Stress Carvalho, D.G.1,*, Kenski, J.C.N.2,*, Rajão, M.A.1, Viola, J.P.B.1, Peeper, D.S.2, Possik, P.A.1 1Brazilian National Cancer Institute, Rio de Janeiro, Brazil; 2The Netherlands Cancer Institute, Amsterdam, The Netherlands *These authors contributed equally to this work BRAF mutations are frequent in melanoma and contribute to disease progression. BRAF inhibitor (BRAFi) treatment leads to rapid regression of melanoma metastases. However, therapy resistance is imminent and represents an important clinical challenge, underscoring the need for new therapeutic targets. Checkpoint kinase 1 (Chk1) is essential in cell cycle regulation and DNA damage response signaling, and is currently explored as a potential target in cancer. Here, we investigated Chk1 as a therapeutic target in BRAF inhibitor-resistant (BRAFiR) melanomas using GDC0575, a specific Chk1 inhibitor (Chk1i). We demonstrated that BRAFiR cells are hypersensitive to Chk1i compared to their respective treatment-naïve melanoma cells. The monitoring of cell cycle progression using a fluorescent cell cycle indicator system showed that a high percentage of BRAFiR cells are unable to complete cell division upon Chk1 inhibition. These data also indicated that S phase progression is crucial for Chk1i-induced cytotoxicity. Bromodeoxyuridine staining followed by flow cytometry showed that Chk1i-treated BRAFiR cells partially failed to incorporate nucleotides during S phase, whereas this effect was minimum in treatment-naïve cells. However, DNA fibers assay revealed decreased fork speed rate for both treatment-naïve and BRAFiR cells upon Chk1i treatment. Finally, we observed that Chk1i induced a greater increase in pRPA and yH2AX levels in BRAFiR cells than in treatment-naïve melanoma cells. Although Chk1i induces replication stress in both BRAFiR and treatment-naïve, we identified that these effects are more pronounced in BRAFiR cells, which accumulated higher levels of DNA damage, suggesting that these cells die after failing to recover from Chk1i-induced replication stress. These observations may help to identify biomarkers for Chk1i sensitivity and contribute to the development of more efficient therapeutic approaches for BRAFiR melanomas. Supported by: MS/INCA, CNPq, ICGEB, FAPERJ

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Impact and consequences of DNA damageon skeletal muscle cells

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Impact and consequences of DNA damageon skeletal muscle cells Haser Hasan SUTCU (1,2*), Miria RICCHETTI (2) and Celine BALDEYRON (1) (1) Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Fontenay-aux-Roses (2) Institut Pasteur, Team Stability of Nuclear and Mitochondrial DNA, Paris (*) previous address DNA double-strand breaks (DSBs) are dangerous DNA damages and a risk factor for genome stability. The maintenance of genome integrity is crucial for adult stem cells that are responsible for regeneration of damaged tissues and tissue homeostasis throughout life. Skeletal muscle regeneration in the adult relies on muscle stem cells (satellite cells, SCs) that display a remarkable DSB repair activity, but the underlying mechanism is poorly elucidated. In a previous study during my PhD (to be submitted) I investigated the impact of impaired DSB repair on skeletal muscle stem cell viability and differentiation. This work also assessed DSB repair mechanism(s) in muscle stem cells and the consequences on muscle regeneration. This study revealed that DSB repair factors affect myogenesis independently of their DNA repair activity, suggesting that this novel function significantly affects muscle regeneration. In particular, this study addressed the role of DNA-dependent protein kinase (DNA-PK), a crucial factor of non-homologous end-joining (NHEJ), a major pathway involved in DSB repair, in muscle differentiation in the mouse. In my present project at the Institute of Radioprotection and Nuclear Safety (IRSN), we are investigating DNA damage and repair not only in satellite cells but also in post-mitotic, multinucleated muscle fibers (myofibers). Despite that myofibers rarely develop into tumour cells, there has been implications on muscle function upon radiotherapy for other cancer tissues. Accordingly, the present project aims to 1) investigate the consequences of reversible/irreversible DNA damage on nuclei of multi-nucleated fibers, 2) understand radiation-induced DNA damage repair mechanisms in myofibers, and 3) assess the impact of DNA damage on the function and viability of the muscle fibers.

Speaker(s):
  • Haser H. Sutcu, PhD, Institut de Radioprotection et de Sûreté Nucléaire (IRSN)

The Use of Microhomologies by RAD51 Generates Genomic Rearrangements Between Non-homologous Sequences

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Function of human DNA polymerase lambda The repair of DNA double-strand breaks (DSBs) is essential for genomic stability but also generates genomic instability: gross chromosome rearrangements (GCR) and/or mutagenesis at the repair junction lead(s) to sequence alterations like, mutations, translocations, deletions or insertions. Analyzing GCR junctions is very informative on the molecular mechanisms that actively generate GCRs. Using intrachromosomal reporters in immortalized fibroblasts, we have shown that repair of distant DSBs, but not close DSBs, favor insertion of ectopic chromosomal sequences at the scar (Guirouilh-Barbat et al. PLOS genetics 2016). These Templated Sequence Insertions (TSIs) are coupled to CtIP-mediated resection and are inhibited by 53BP1. Here we show that insertions of TSIs are observed in a physio-pathological context at junctions of rearrangements implicating distant ends, but not close ends, in breast tumors. In addition, we found TSIs to be less frequent in BRCA2 deficient tumors and we confirmed the implication of BRCA2 and RAD51 in the capture of TSIs using our intrachromosomal reporter for distant DSB repair in fibroblasts. High Throughput Genome wide Translocations Sequencing (HTGTS) revealed that i) RAD51 mediates complex genomic rearrangements that are observed upon 53BP1 depletion, ii) RAD51 is implicated in bipartite rearrangements, i.e. translocations. Mimicking the reciprocal hNMP1-ALK translocation with CRIPR-Cas9 in RPE1 cells confirmed that RAD51 mediates the increased frequency of translocation in 53BP1 depleted cells. None of these RAD51-mediated genomic rearrangements (translocations, complex rearrangements, capture of TSIs) involve sequence homologies, but only microhomologies. Using transmission electron microscopy (TEM) we found that RAD51 can foster the pairing of heterologous molecules using one or two microhomology patch(es). Our data show that RAD51 and BRCA2 have the intrinsic capacity to generate genetic instability through a mechanism independent of sequence homology and that this capacity is counteracted by 53BP1. We propose that these events arise by microhomology-mediated template switch when RAD51 samples DNA in the course of the search of homology .

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A meta-analysis of clinical cases of reversion mutations in BRCA genes identifies signatures of DNA end-joining repair mechanisms driving therapy resistance

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

A meta-analysis of clinical cases of reversion mutations in BRCA genes identifies signatures of DNA end-joining repair mechanisms driving therapy resistance L. Tobalina1, J. Armenia1, E. Irving2, M. J. O’Connor2 and J. V. Forment2 1Bioinformatics and data science and 2DDR Biology, Bioscience; Oncology R&D, AstraZeneca, Cambridge, UK Background Germline mutations in the BRCA1 or BRCA2 (BRCA) genes predispose to hereditary breast and ovarian cancer and, mostly in the case of BRCA2, are also prevalent in cases of pancreatic and prostate malignancies. Tumours from these patients tend to lose both copies of the wild type BRCA gene, which makes them exquisitely sensitive to platinum drugs and PARP inhibitors (PARPi), treatments of choice in these disease settings. Reversion secondary mutations with the capacity of restoring BRCA protein expression have been documented in the literature as bona fide mechanisms of resistance to these treatments. Patients We analyse published sequencing data of BRCA genes (from tumour or circulating tumour DNA) in 327 patients with tumours harbouring mutations in BRCA1 or BRCA2 (234 patients with ovarian cancer, 27 with breast cancer, 13 with pancreatic cancer, 11 with prostate cancer and 42 with a cancer of unknown origin) that progressed on platinum or PARPi treatment. Results We describe 269 cases of reversion mutations in 86 patients in this cohort (26.3%). Detailed analyses of the reversion events underlines the different importance of BRCA protein domains in contributing to resistance, as revertant proteins can be devoid of sizable parts of the original sequence. They also highlight the key role of mutagenic end-joining DNA repair pathways in generating reversions, especially in those affecting BRCA2. Conclusions Our analyses suggest that pharmacological inhibition of DNA end-joining repair pathways could improve durability of drug treatments by preventing the acquisition of reversion mutations in BRCA genes. They also highlight potential new therapeutic opportunities when reversions result in expression of hypomorphic versions of BRCA proteins, especially with agents targeting the response to DNA replication stress.

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TICRR/TRESLIN protein expression is cell cycle regulated by the CUL4-DDB1 E3 Ligase

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

TICRR/TRESLIN protein expression is cell cycle regulated by the CUL4-DDB1 E3 Ligase Kimberlie A. Wittig1,2, Courtney G. Sansam2, Tyler D. Noble1,2, Duane Goins2, Christopher L. Sansam1,2* 1 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA; 2 Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA A DNA replication program, which ensures that the genome is accurately and completely replicated, is established during G1 prior to the onset of S-phase. Tight regulation of the number of active replisomes is crucial to prevent replication stress-induced DNA damage. TICRR/TRESLIN is essential for DNA replication initiation, and the expression level of TICRR and its phosphorylation determine the number of origins that initiate simultaneously during S-phase. However, the mechanisms regulating TICRR protein expression are unknown. Here, we aimed to evaluate TICRR protein dynamics around the G1/S-phase transition. We tagged the endogenous C-terminus of TICRR with mClover in HCT-116 cells using CRISPR/Cas9 and applied an established flow cytometry assay to detect how levels of both insoluble and total TICRR change. Total TICRR expression is highest in G2/M, decreases with cell division, and further decreases at the G1/S-phase transition. However, insoluble TICRR levels are highest in G1 and sharply decrease with S-phase entry. Although total TICRR expression decreases between G2/M and G1, insoluble TICRR levels increase demonstrating that insoluble TICRR accumulation in G1 is not due to changes in its expression. In contrast, both total and insoluble TICRR levels decrease with S-phase entry demonstrating this decrease is at least in part due to the degradation of TICRR protein. Utilizing proteasomal and neddylation inhibitors, we show that degradation of TICRR depends on Cullin E3 ligases and is specific to S-phase. Additionally, through a targeted siRNA screen we have identified CUL4-DDB1 as the Cullin complex necessary for TICRR degradation. Collectively, our results demonstrate how total and insoluble levels of TICRR change in distinct ways throughout the cell cycle, and we have elucidated a mechanism for TICRR degradation at the G1/S transition affecting the levels of protein available for DNA replication initiation during S-phase. These results suggest a mechanism to control the rate of origin firing to prevent replication stress and DNA damage.

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Mechanism of genomic rearrangements driven by NAHR in response to replication stress

Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Mechanism of genomic rearrangements driven by NAHR in response to replication stress Repeated sequences, which are very common in genomes, provide additional opportunities for non-allelic homologous recombination (NAHR). These events can lead to genetic alterations such as deletions, inversions, translocations or copy number variation. As a consequence, many human disorders result from genomic rearrangements involving repeated sequences. For example, the Alu repeats, which are present in ~1 million copies in the human genome, have been found at the deletion breakpoints of a considerable number of genes involved in human pathologies. Despite extensive observations that repeated sequences are predisposed to rearrangements, the events leading to these reactions are not fully understood. Here, we explored the possibility that replication stress, a typical threat to cell survival, could trigger recombination between repeats, and identified the genes regulating this process. In order to detect and study recombination between repeated sequences, we examined ADE2 recombinants in the yeast genome, which are generated by recombination between inverted repeats of two ade2 mutant alleles separated by a 1.3kb TRP1 gene. It was published previously that spontaneous recombination between the two ade2 copies is highly dependent on Rad52, acting in two independent recombination pathways. First, Rad52 acts as a loader of Rad51 which mediates gene conversion events. In the second pathway, Rad52, in association with Rad59, promotes inversion events which reverse the intervening TRP1 sequence. We show that NAHR between the two ade2 copies is greatly stimulated by a localized replication barrier (Tus/Ter system) at the precise site of the inverted repeats. Thus, our results strongly suggest that fork stalling near repeated sequences can stimulate NAHR. Unlike spontaneous events, our genetic study reveals that replication associated NAHR reactions rely on a unique and particular pathway dependent on Rad51 catalytic activity, Rad52 and its single-strand annealing partner Rad59. We propose a model implicating a template switch event between the repeated sequences on the two sister chromatids at a stalled replication fork. Our genetic observations suggest that following fork stalling, the fork reversal proteins Rad5 and Mph1, together with the DNA end resection nucleases Mre11 and Exo1, create a ssDNA substrate onto which the Rad57-Rad55 paralogs and the Shu complex facilitate Rad51 filament assembly. The recombinase then catalyses strand invasion of the parental duplex on one of the two repeats, which initiates DNA synthesis by the replicative DNA polymerase Pol δ. Depending on how long DNA synthesis is, the recombination product after resolution of the reversed fork can be gene conversion or inversion.

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