New tools to study DNA double-strand break repair pathway choice

A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB) repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences

EXOSC10 is required for RPA assembly and controlled DNA end resection at DNA double-strand breaks

The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism. Here we show that the exosome is necessary for the repair of DNA double-strand breaks (DSBs) in human cells and that RNA clearance is an essential step in homologous recombination. Transcription of DSB-flanking sequences results in the production of damage-induced long non-coding RNAs (dilncRNAs) that engage in DNA-RNA hybrid formation. Depletion of EXOSC10, an exosome catalytic subunit, leads to increased dilncRNA and DNA-RNA hybrid levels. Moreover, the targeting of the ssDNA-binding protein RPA to sites of DNA damage is impaired whereas DNA end resection is hyper-stimulated in EXOSC10-depleted cells. The DNA end resection deregulation is abolished by transcription inhibitors, and RNase H1 overexpression restores the RPA recruitment defect caused by EXOSC10 depletion, which suggests that RNA clearance of newly synthesized dilncRNAs is required for RPA recruitment, controlled DNA end resection and assembly of the homologous recombination machinery.España, Ministerio de Economía y Competitividad R + D + I project grant SAF2016-74855-P to P.

Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation

THO/TREX is a conserved eukaryotic complex formed by the core THO complex plus proteins involved in mRNA metabolism and export such as Sub2 and Yra1. Mutations in any of the THO/TREX structural genes cause pleiotropic phenotypes such as transcription impairment, increased transcription-associated recombination, and mRNA export defects. To assay the relevance of THO/TREX complex in transcription, we performed in vitro transcription elongation assays in mutant cell extracts using supercoiled DNA templates containing two G-less cassettes. With these assays, we demonstrate that hpr1Δ, tho2Δ, and mft1Δ mutants of the THO complex and sub2 mutants show significant reductions in the efficiency of transcription elongation. The mRNA expression defect of hpr1Δ mutants was not due to an increase in mRNA decay, as determined by mRNA half-life measurements and mRNA time course accumulation experiments in the absence of Rrp6p exoribonuclease. This work demonstrates that THO and Sub2 are required for efficient transcription elongation, providing further evidence for the coupling between transcription and mRNA metabolism and export.Ministerio de Ciencia y Tecnología BMC2000-0439Human Frontier Science Program RG1999/007

Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice

DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks.España , Ministerio de Economía y Competitividad SAF2010-1487

ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.This work was funded by an R + D + I grant from the Agencia Estatal de Investigación PID2019-104195GB-I00/ AEI/10.13039/501100011033 (P.H.), R + D + I grants from the Spanish Ministry of Economy and Competitiveness (SAF2016-74855-P to P.H. and BFU2016-75058-P to A.A.), the European Research Council (ERC2014 AdG669898 TARLOOP to A.A.), the Associazione Italiana Ricerca sul Cancro-AIRC (AIRC IG 22080 to A.G.), The Swedish Research Council (grants 2015-04553 to N.V.), The Swedish Cancer Society (grant CAN 2016/460 to N.V.) and the European Union (FEDER). AR is, and FM-N and RP-C were, funded with FPU fellowships from the Spanish Ministry of Education and S.S. by a Juan de la Cierva contract from the Spanish Ministry of Economy and Competitiveness. J.D.P. and M.E-C. were supported by the Department of Molecular Biosciences, The Wenner-Gren Institute at Stockholm University. J.D-P. and R.P-C. were recipients of short-term EMBO fellowships (STF- 7513-2018 and STF-7764-2018). The mutational results shown here are in whole or part based upon the data generated by the TCGA Research Network: https://www.cancer.gov/tcga. All the bioinformatic analyses were performed using custom scripts that were run on the High performance computing cluster provided by the Centro Informático Cienti ́fico de Andalucía (CICA). CABIMER is supported by the regional government of Andalucia (Junta de Andalucía)

143 PRINCIPAL COMPONENT CLUSTERING OF FRONTAL PLANE KNEE KINEMATICS

Replication forks stall at different DNA obstacles such as those originated by transcription. Fork stalling can lead to DNA double-strand breaks (DSBs) that will be preferentially repaired by homologous recombination when the sister chromatid is available. The Rrm3 helicase is a replisome component that promotes replication upon fork stalling, accumulates at highly transcribed regions and prevents not only transcription-induced replication fork stalling but also transcription-associated hyper-recombination. This led us to explore the possible role of Rrm3 in the repair of DSBs when originating at the passage of the replication fork. Using a mini-HO system that induces mainly single-stranded DNA breaks, we show that rrm3Δ cells are defective in DSB repair. The defect is clearly seen in sister chromatid recombination, the major repair pathway of replication-born DSBs. Our results indicate that Rrm3 recruitment to replication-born DSBs is crucial for viability, uncovering a new role for Rrm3 in the repair of broken replication forks.This work was supported by grants from the Spanish Ministry of Economy and Innovation (BFU2013-42918), the European Union (FEDER), the European Research Council (ERC2014 AdG669898 TARLOOP), and the Junta de Andalucía (BIO1238). SMG was funded by a predoctoral training fellowship from the Spanish National Research Council (CSIC) and BGG by a postdoctoral grant from the Scientific Foundation of the Spanish Association Against Cancer (AECC). Funding for open access charge: Grants from the Spanish Ministry of Economy and Innovation (BFU2013-42918).Peer Reviewe

The Helicase PIF1 Facilitates Resection over Sequences Prone to Forming G4 Structures

DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpressio

Quantitative muscle MRI to follow up late onset Pompe patients : a prospective study

Late onset Pompe disease (LOPD) is a slow, progressive disorder characterized by skeletal and respiratory muscle weakness. Enzyme replacement therapy (ERT) slows down the progression of muscle symptoms. Reliable biomarkers are needed to follow up ERT-treated and asymptomatic LOPD patients in clinical practice. In this study, 32 LOPD patients (22 symptomatic and 10 asymptomatic) underwent muscle MRI using 3-point Dixon and were evaluated at the time of the MRI with several motor function tests and patient-reported outcome measures, and again after one year. Muscle MRI showed a significant increase of 1.7% in the fat content of the thigh muscles in symptomatic LOPD patients. In contrast, there were no noteworthy differences between muscle function tests in the same period of time. We did not observe any significant changes either in muscle MRI or in muscle function tests in asymptomatic patients over the year. We conclude that 3-point Dixon muscle MRI is a useful tool for detecting changes in muscle structure in symptomatic LOPD patients and could become part of the current follow-up protocol in daily clinics

Follow-up of late-onset Pompe disease patients with muscle magnetic resonance imaging reveals increase in fat replacement in skeletal muscles

Altres ajuts: This investigation was sponsored by the following grants, one from Sanofi Genzyme and another from the Spanish Ministry of Health, Fondos FEDER-ISCIII. Isabel Illa has received speaker honorarium from Grifols and Sanofi-Genzyme. Jordi Díaz-Manera has received speaker honorarium from PTC Therapeutics and Sanofi-Genzyme. The authors of this manuscript certify that they comply with the ethical guidelines for authorship and publishing in the Journal of Cachexia, Sarcopenia, and Muscle.42Late-onset Pompe disease (LOPD) is a genetic disorder characterized by progressive degeneration of the skeletal muscles produced by a deficiency of the enzyme acid alpha-glucosidase. Enzymatic replacement therapy with recombinant human alpha-glucosidase seems to reduce the progression of the disease; although at the moment, it is not completely clear to what extent. Quantitative muscle magnetic resonance imaging (qMRI) is a good biomarker for the follow-up of fat replacement in neuromuscular disorders. The aim of this study was to describe the changes observed in fat replacement in skeletal muscles using qMRI in a cohort of LOPD patients followed prospectively. A total of 36 LOPD patients were seen once every year for 4 years. qMRI, several muscle function tests, spirometry, activities of daily living scales, and quality-of-life scales were performed on each visit. Muscle MRI consisted of two-point Dixon studies of the trunk and thigh muscles. Computer analysis of the images provided the percentage of muscle degenerated and replaced by fat in every muscle (known as fat fraction). Longitudinal analysis of the measures was performed using linear mixed models applying the Greenhouse-Geisser test. We detected a statistically significant and continuous increase in mean thigh fat fraction both in treated (+5.8% in 3 years) and in pre-symptomatic patients (+2.6% in 3years) (Greenhouse-Geisser p < 0.05). As an average, fat fraction increased by 1.9% per year in treated patients, compared with 0.8% in pre-symptomatic patients. Fat fraction significantly increased in every muscle of the thighs. We observed a significant correlation between changes observed in fat fraction in qMRI and changes observed in the results of the muscle function tests performed. Moreover, we identified that muscle performance and mean thigh fat fraction at baseline visit were independent parameters influencing fat fraction progression over 4 years (analysis of covariance, p < 0.05). Our study identifies that skeletal muscle fat fraction continues to increase in patients with LOPD despite the treatment with enzymatic replacement therapy. These results suggest that the process of muscle degeneration is not stopped by the treatment and could impact muscle function over the years. Hereby, we show that fat fraction along with muscle function tests can be considered a good outcome measures for clinical trials in LOPD patients

Sem1 is a functional component of the nuclear pore complex–associated messenger RNA export machinery

The evolutionarily conserved protein Sem1/Dss1 is a subunit of the regulatory particle (RP) of the proteasome, and, in mammalian cells, binds the tumor suppressor protein BRCA2. Here, we describe a new function for yeast Sem1. We show that sem1 mutants are impaired in messenger RNA (mRNA) export and transcription elongation, and induce strong transcription-associated hyper-recombination phenotypes. Importantly, Sem1, independent of the RP, is functionally linked to the mRNA export pathway. Biochemical analyses revealed that, in addition to the RP, Sem1 coenriches with components of two other multisubunit complexes: the nuclear pore complex (NPC)-associated TREX-2 complex that is required for transcription-coupled mRNA export, and the COP9 signalosome, which is involved in deneddylation. Notably, targeting of Thp1, a TREX-2 component, to the NPC is perturbed in a sem1 mutant. These findings reveal an unexpected nonproteasomal function of Sem1 in mRNA export and in prevention of transcription-associated genome instability. Thus, Sem1 is a versatile protein that might stabilize multiple protein complexes involved in diverse pathways.España, Ministerio de Educación y Ciencia BFU2006-05260 and BFU2007-28647Andalucia, Junta de Andalucia CVI102 and CVI254