Studying DNA Double-Strand Break Repair: An Ever-Growing Toolbox

To ward off against the catastrophic consequences of persistent DNA double-strand breaks (DSBs), eukaryotic cells have developed a set of complex signaling networks that detect these DNA lesions, orchestrate cell cycle checkpoints and ultimately lead to their repair. Collectively, these signaling networks comprise the DNA damage response (DDR). The current knowledge of the molecular determinants and mechanistic details of the DDR owes greatly to the continuous development of ground-breaking experimental tools that couple the controlled induction of DSBs at distinct genomic positions with assays and reporters to investigate DNA repair pathways, their impact on other DNA-templated processes and the specific contribution of the chromatin environment. In this review, we present these tools, discuss their pros and cons and illustrate their contribution to our current understanding of the DDR.European Research Council (ERC-2014-CoG 647344

Live-cell imaging of transcriptional activity at DNA double-strand breaks

Copyright © 2021 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported LicenseDNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far elusive how DSBs affect transcription. A reason for this was the lack of suitable tools to simultaneously monitor transcription and the induction of a genic DSB with sufficient temporal and spatial resolution. This work describes a set of new reporters that directly visualize transcription in live cells immediately after the induction of a DSB in the DNA template. Bacteriophage RNA stem-loops are employed to monitor the transcription with single-molecule sensitivity. For targetting the DSB to a specific gene region, the reporter genes are engineered to contain a single recognition sequence of the homing endonuclease I-SceI, otherwise absent from the human genome. A single copy of each reporter gene was integrated into the genome of human cell lines. This experimental system allows the detection of single RNA molecules generated by the canonical gene transcription or by DNA break-induced transcription initiation. These reporters provide an unprecedented opportunity for interpreting the reciprocal interactions between transcription and DNA damage and to disclose hitherto unappreciated aspects of DNA break-induced transcription.This work was funded by PTDC/MED-OUT/32271/2017, PTDC/BIA-MOL/30438/2017 and PTDC/MED-OUT/4301/2020 from Fundação para a Ciência e a Tecnologia (FCT), Portugal and by LISBOA-01-0145-FEDER-007391, project cofunded by FEDER through POR Lisboa, Portugal 2020-Programa Operacional Regional de Lisboa, and FCT. Funding was also received from EU Horizon 2020 Research and Innovation Programme (RiboMed 857119). M.A. is the recipient of the FCT Ph.D. fellowship 2020.05899.BD.info:eu-repo/semantics/publishedVersio

A link between nuclear RNA surveillance, the human exosome and RNA polymerase II transcriptional termination

In eukaryotes, the production of mature messenger RNA that exits the nucleus to be translated into protein in the cytoplasm requires precise and extensive modification of the nascent transcript. Any failure that compromises the integrity of an mRNA may cause its retention in the nucleus and trigger its degradation. Multiple studies indicate that mRNAs with processing defects accumulate in nuclear foci or ‘dots’ located near the site of transcription, but how exactly are defective RNAs recognized and tethered is still unknown. Here, we present evidence suggesting that unprocessed β-globin transcripts render RNA polymerase II (Pol II) incompetent for termination and that this quality control process requires the integrity of the nuclear exosome. Our results show that unprocessed pre-mRNAs remain tethered to the DNA template in association with Pol II, in an Rrp6-dependent manner. This reveals an unprecedented link between nuclear RNA surveillance, the exosome and Pol II transcriptional termination

Methyl 2-(4,6-dichloro-1,3,5-triazin-2-yl­amino)acetate

The title compound, C6H6Cl2N4O2, was prepared by the nucleophilic substitution of 2,4,6-trichloro-1,3,5-triazine by glycine methyl ester hydro­chloride, and was isolated from the reaction by using flash chromatography. The crystal structure at 150 K reveals the presence two crystallographically independent mol­ecules in the asymmetric unit which differ in the orientation of the pendant methoxy­carbonyl group. Each mol­ecular unit is engaged in strong and highly directional N—H⋯N hydrogen-bonding inter­actions with a symmetry-related mol­ecule, forming supra­molecular dimers which act as the synthons in the crystal packing

Surgical Myocardial Revascularization of Patients with Ischemic Cardiomyopathy and Severe Left Ventricular Disfunction

OBJECTIVE: To determine long-term survival, identify preoperative factors predictive of a favorable outcome, and assess functional improvement after coronary artery bypass grafting in patients with advanced left ventricular dysfunction. METHODS: Between 1995 and 2001, 244 patients who underwent coronary artery bypass grafting and had a preoperative left ventricular ejection fraction less than or equal to 35% were included. left ventricular ejection fraction was determined by uniplanar or biplanar ventriculography during left heart catheterization. Indication for surgery was predominance of tissue viability. Functional improvement was evaluated through echocardiography and gated scintigraphy at exercise/ rest. Survival was determined by Kaplan-Meier analysis. RESULTS: Mean left ventricular ejection fraction was 29±4% (ranged from 9% to 35%). An average of 3.01 coronary bypass grafts per patient were performed. In-hospital mortality was 3.7% (9 patients). The 4-year survival rate was 89.7%. Multivariate correlates of favorable short- and long-term outcome were preoperative New York Heart Association Funcional classification for congestive heart failure class I/II, lower PAsP, higher left ventricular ejection fraction and gated left ventricular ejection fraction Ex/Rest ratio >5%. Left ventricular ejection fraction rise from 32±5% to 39±5%, p <0.001. Gated left ventricular ejection fraction at exercise/ rest increased markedly after surgery: from 27±8%/ 23±7% to 37±5%/ 31±6%, p <0.001. CONCLUSIONS: In selected patients with severe ischemic left ventricular dysfunction and predominance of tissue viability, coronary artery bypass grafting may be capable of implement preoperative clinical/ functional parameters in predicting outcome as left ventricular ejection fraction and gated left ventricular ejection fraction at exercise/ rest

Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1

© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.Ataxia telangiectasia mutated and Rad3-related (ATR) kinase is a key factor activated by DNA damage and replication stress. An alternative pathway for ATR activation has been proposed to occur via stalled RNA polymerase II (RNAPII). However, how RNAPII might signal to activate ATR remains unknown. Here, we show that ATR signaling is increased after depletion of the RNAPII phosphatase PNUTS-PP1, which dephosphorylates RNAPII in its carboxy-terminal domain (CTD). High ATR signaling was observed in the absence and presence of ionizing radiation, replication stress and even in G1, but did not correlate with DNA damage or RPA chromatin loading. R-loops were enhanced, but overexpression of EGFP-RNaseH1 only slightly reduced ATR signaling after PNUTS depletion. However, CDC73, which interacted with RNAPII in a phospho-CTD dependent manner, was required for the high ATR signaling, R-loop formation and for activation of the endogenous G2 checkpoint after depletion of PNUTS. In addition, ATR, RNAPII and CDC73 co-immunoprecipitated. Our results suggest a novel pathway involving RNAPII, CDC73 and PNUTS-PP1 in ATR signaling and give new insight into the diverse functions of ATR.Norwegian Cancer Society [3367910]; South-Eastern Norway Health Authorities [2014035, 2013017]; Norwegian Research Council [275918]; EEA Czech-Norwegian Research Programme (Norwegian Financial Mechanism 2009–2014 and the Ministry of Education, Youth and Sports [Project Contract no. MSMT-22477/2014 (7F14061)]. Funding for open access charge: Norwegian Research Council.info:eu-repo/semantics/publishedVersio

Glycine methyl ester hydro­chloride

The title compound [systematic name: (methoxy­carbonyl­meth­yl)ammonium chloride], crystallizes as a salt, C3H8NO2 +·Cl−, with the charged species inter­acting mutually via strong and highly directional N+—H⋯Cl− hydrogen bonds which lead to the formation of a supra­molecular tape running parallel to the c axis. Tapes close pack in the solid state mediated by multipoint recognition synthons based on weak C—H⋯O inter­actions and van der Waals contacts between adjacent methyl groups

T cell activation regulates CD6 alternative splicing by transcription dynamics and SRSF1

The T cell-surface glycoprotein CD6 is a modulator of cellular responses and has been implicated in several autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis. During Ag presentation, CD6 is targeted to the immunological synapse in a ligand binding-dependent manner, in which CD6 domain 3 directly contacts CD166, expressed on the APC. T cell activation results in the induction of CD6?d3, an alternatively spliced isoform that lacks the ligand-binding domain and thus no longer localizes at the immunological synapse. In this study, we investigated the molecular mechanisms regulating the expression of CD6?d3 upon human primary T cell activation. Using chromatin immunoprecipitation, we observed an increase in RNA polymerase II occupancy along the CD6 gene and augmented CD6 transcription. We showed that activation leads to transcription-related chromatin modifications, revealed by higher CD6 acetylation levels. Modulation of chromatin conformation using a histone deacetylase inhibitor that increases transcription rate causes an increase of exon 5 skipping. We further showed that the splicing factor SRSF1 binds to a regulatory element in CD6 intron 4, activating exon 5 splicing and promoting exon 5 inclusion. Concomitant with T cell activation-induced exon 5 skipping, we observed a downregulation of SRSF1. Using RNA immunoprecipitation, we showed that in activated T cells, SRSF1 recruitment to the CD6 transcript is impaired by increased chromatin acetylation levels. We propose that upon T cell activation, SRSF1 becomes limiting, and its function in CD6 exon 5 splicing is countered by an increase in CD6 transcription, dependent on chromatin acetylation. Copyright © 2014 by The American Association of Immunologists, Inc.This work was supported by the European Regional Development Fund, Programa Operacional Ciencia, Tecnologia e Inovacao 2010 (POCI and POCTI 2010), and the Fundacao para a Ciencia e Tecnologia (PTDC/SAU-GMG/116621/2010, PTDC/BEX-BCM/0468/2012, PTDC/IMI-IMU/0158/2012)

Resistividade em solos: efeito dos índices físicos e condições de análise / Soil resistivity: phisical indexes and analysis conditions influence

A resistividade elétrica é um parâmetro amplamente utilizado na avaliação da corrosividade dos solos. Entretanto, algumas metodologias de análise em laboratório têm divergência nos procedimentos e resultados obtidos, o que pode impactar na correta avaliação da propriedade e sua consequente correlação com a resistência à corrosão. Neste estudo foi realizado uma revisão dos ensaios de resistividade de solo propostos pelos procedimentos adotados pelas normas ABNT NBR 16254-1:2014 (Anexo C), ASTM G-187-15 e ASTM G-187-15 modificada pela adoção de cálculos dos índices físicos para obtenção do grau de saturação da amostra de solo. A avaliação dos resultados indicou que o os procedimentos realizados pela norma ASTM-G187-15 e pelo mesmo procedimento modificado, adotando os índices físicos foram compatíveis. Já o ensaio realizado pela norma ABNT NBR 16254-1:2014 (Anexo C) não teve resultados satisfatórios