DNA Double Strand Breaks and Chromosomal Translocations Induced by DNA Topoisomerase II

DNA double strand breaks (DSBs) are the most cytotoxic lesions of those occurring in the DNA and can lead to cell death or result in genome mutagenesis and chromosomal translocations. Although most of these rearrangements have detrimental effects for cellular survival, single events can provide clonal advantage and result in abnormal cellular proliferation and cancer. The origin and the environment of the DNA break or the repair pathway are key factors that influence the frequency at which these events appear. However, the molecular mechanisms that underlie the formation of chromosomal translocations remain unclear. DNA topoisomerases are essential enzymes present in all cellular organisms with critical roles in DNA metabolism and that have been linked to the formation of deleterious DSBs for a long time. DSBs induced by the abortive activity of DNA topoisomerase II (TOP2) are “trending topic” because of their possible role in genome instability and oncogenesis. Furthermore, transcription associated TOP2 activity appears to be one of the most determining causes behind the formation of chromosomal translocations. In this review, the origin of recombinogenic TOP2 breaks and the determinants behind their tendency to translocate will be summarized.Spanish Ministry of Science and Universities BFU2016-76446-P, RYC-2014-1666

DNA Double Strand Breaks and Chromosomal Translocations Induced by DNA Topoisomerase II

Copyright © 2019 Gómez-HerrerosDNA double strand breaks (DSBs) are the most cytotoxic lesions of those occurring in the DNA and can lead to cell death or result in genome mutagenesis and chromosomal translocations. Although most of these rearrangements have detrimental effects for cellular survival, single events can provide clonal advantage and result in abnormal cellular proliferation and cancer. The origin and the environment of the DNA break or the repair pathway are key factors that influence the frequency at which these events appear. However, the molecular mechanisms that underlie the formation of chromosomal translocations remain unclear. DNA topoisomerases are essential enzymes present in all cellular organisms with critical roles in DNA metabolism and that have been linked to the formation of deleterious DSBs for a long time. DSBs induced by the abortive activity of DNA topoisomerase II (TOP2) are “trending topic” because of their possible role in genome instability and oncogenesis. Furthermore, transcription associated TOP2 activity appears to be one of the most determining causes behind the formation of chromosomal translocations. In this review, the origin of recombinogenic TOP2 breaks and the determinants behind their tendency to translocate will be summarized.FG-H laboratory is funded by the Spanish Ministry of Science and Universities (BFU2016-76446-P, RYC-2014-16665

TFIIS is required for the balanced expression of the genes encoding ribosomal components under transcriptional stress

Transcription factor IIS (TFIIS) stimulates RNA cleavage by RNA polymerase II by allowing backtracked enzymes to resume transcription elongation. Yeast cells do not require TFIIS for viability, unless they suffer severe transcriptional stress due to NTP-depleting drugs like 6-azauracil or mycophenolic acid. In order to broaden our knowledge on the role of TFIIS under transcriptional stress, we carried out a genetic screening for suppressors of TFIIS-lacking cells’ sensitivity to 6-azauracil and mycophenolic acid. Five suppressors were identified, four of which were related to the transcriptional regulation of those genes encoding ribosomal components [rRNAs and ribosomal proteins (RP)], including global regulator SFP1. This led us to discover that RNA polymerase II is hypersensitive to the absence of TFIIS under NTP scarcity conditions when transcribing RP genes. The absence of Sfp1 led to a profound alteration of the transcriptional response to NTP-depletion, thus allowing the expression of RP genes to resist these stressful conditions in the absence of TFIIS. We discuss the effect of transcriptional stress on ribosome biogenesis and propose that TFIIS contributes to prevent a transcriptional imbalance between rDNA and RP genes.España Ministerio de Economía y competitividad BFU2007-67575-C03-02España Ministerio de Economía y competitividad BFU-2010-21975-C03-03Andalucía, Junta de Andalucía P07-CVI-02623Andalucía, Junta de Andalucía P08-CVI-035

The ribosome assembly gene network is controlled by the feedback regulation of transcription elongation

Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion conditions, Spt6 was physically targeted to the upregulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesismutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes.Ministerio de Economía y Competitividad BFU2013-48643-C3-1-P, BFU2016-77728-C3-1-P, BFU2013-48643-C3- 3-P, BFU2013-42958-PJunta de Andalucía P12-BIO1938MO, P08-CVI-03508Comunidad Valenciana 2015/00

One step back before moving forward: regulation of transcription elongation by arrest and backtracking

RNA polymerase II backtracking is a well-known phenomenon, but its involvement in gene regulation is yet to be addressed. Structural studies into the backtracked complex, new reactivation mechanisms and genome-wide approaches are shedding some light on this interesting aspect of gene transcription. In this review, we briefly summarise these new findings, comment about some results recently obtained in our laboratory, and propose a new model for the influence of the chromatin context on RNA polymerase II backtracking.Ministerio de Economía y Competitividad de España. BFU2007-67575-C03-02 y BFU-2010-21975-C03-03Junta de Andalucía. P07-CVI-02623 y P08-CVI-0350

TDP2-Dependent Non-Homologous End-Joining Protects against Topoisomerase II-Induced DNA Breaks and Genome Instability in Cells and In Vivo

Anticancer topoisomerase >poisons> exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5′-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.Gobierno Español SAF2010-21017, BFU2010-11042-EEuropean Union PERG07- 2010-268466Queen Elisabeth Medical Foundation GSKE 111

PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage

Amyotrophic lateral sclerosis (ALS) is associated with progressive degeneration of motor neurons. Several of the genes associated with this disease encode proteins involved in RNA processing, including fused-in-sarcoma/translocated-in-sarcoma (FUS/TLS). FUS is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins that bind thousands of pre-mRNAs and can regulate their splicing. Here, we have examined the possibility that FUS is also a component of the cellular response to DNA damage. We show that both GFP-tagged and endogenous FUS re-localize to sites of oxidative DNA damage induced by UVA laser, and that FUS recruitment is greatly reduced or ablated by an inhibitor of poly (ADP-ribose) polymerase activity. Consistent with this, we show that recombinant FUS binds directly to poly (ADP-ribose) in vitro, and that both GFP-tagged and endogenous FUS fail to accumulate at sites of UVA laser induced damage in cells lacking poly (ADP-ribose) polymerase-1. Finally, we show that GFP-FUS(R521G), harbouring a mutation that is associated with ALS, exhibits reduced ability to accumulate at sites of UVA laser-induced DNA damage. Together, these data suggest that FUS is a component of the cellular response to DNA damage, and that defects in this response may contribute to ALS

Genome-scale mapping of DNA damage suppressors through phenotypic CRISPR-Cas9 screens

© 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance.This work was supported by grants from NIH (RM1HG010461 and UM1HG011989, to F.P.R.), from the Canadian Cancer Society (705644 to D.D.), and from the Canadian Institutes of Health Research (CIHR, grants PJT-180438 to D.D. and FDN159926 to F.P.R.).Peer reviewe

Monitorización y seguimiento del esfuerzo realizado por los estudiantes y de su asistencia a actividades presenciales

Este artículo documenta el planteamiento, la metodología y los primeros resultados de un plan de monitorización detallada del esfuerzo y de asistencia a actividades presenciales por parte de los estudiantes de las titulaciones ofertadas por la Escuela Técnica Superior de Ingenieros Navales de la Universidad Politécnica de Madrid durante el segundo cuatrimestre del curso 2011-2012. Se ha establecido un sistema mecánico de recogida de datos de esfuerzo por parte de los estudiantes utilizando una hoja tipo test especialmente configurada al efecto. Se pasa una hoja en todas y cada una de las actividades presenciales realizadas y en la hoja se solicita información sobre el trabajo "fuera de clase". Se documenta en este artículo cómo se ha estructurado esa hoja, qué tipo de datos se recogen, cómo se tratan mediante una base de datos creada al efecto, qué tipo de análisis se puede realizar y qué resultados preliminares obtenemos de dichos análisis