Topoisomerase 2 (TOP2) DNA transactions proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer ...TOP2 drugs. How genotoxic TOP2 DNA-protein cross-links are resolved is unclear. We found that the SUMO (small ubiquitin-related modifier) ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a “split-SIM” SUMO2 engagement platform. These findings uncover a ZATT-TDP2–catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc.
•Human Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after IR-mediated DSBs in vivo.•Polλ Thr204 is the main target for ATM- and DNA-PKcs-mediated ...phosphorylation.•Phosphorylation of Polλ at Thr204 after IR is required for efficient Polλ-mediated gap-filling NHEJ repair of a subset of DSBs in vivo.•Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs.
DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.
Accumulation of topological stress in the form of DNA supercoiling is inherent to the advance of RNA polymerase II (Pol II) and needs to be resolved by DNA topoisomerases to sustain productive ...transcriptional elongation. Topoisomerases are therefore considered positive facilitators of transcription. Here, we show that, in contrast to this general assumption, human topoisomerase IIα (TOP2A) activity at promoters represses transcription of immediate early genes such as c-FOS, maintaining them under basal repressed conditions. Thus, TOP2A inhibition creates a particular topological context that results in rapid release from promoter-proximal pausing and transcriptional upregulation, which mimics the typical bursting behavior of these genes in response to physiological stimulus. We therefore describe the control of promoter-proximal pausing by TOP2A as a layer for the regulation of gene expression, which can act as a molecular switch to rapidly activate transcription, possibly by regulating the accumulation of DNA supercoiling at promoter regions.
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•Catalytic inhibition of TOP2A results in a global reduction in promoter-proximal pausing•Catalytic inhibition of TOP2A upregulates transcription of immediate early genes (IEGs)•IEG upregulation occurs independently of DNA breaks or cellular stress•IEG upregulation depends on the accumulation of negative supercoiling at promoter regions
Herrero-Ruiz et al. analyze the function of DNA topoisomerase II in transcriptional regulation. The results reveal that TOP2A activity favors promoter-proximal pausing of RNA polymerase II and repression of immediate early genes in a mechanism that is independent of DNA break formation and involves the control of DNA supercoiling at promoters.
Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and ...misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.
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•Chaperone proteins interact with RNA in human cells•HSP70 binds to non-coding RNA•HSP70 occupies RNA polymerase III-transcribed genes•HSP70 regulates RNA polymerase III transcription
Leone and Srivastava et al. describe the direct interaction of chaperone proteins with RNA in human cells. They describe the localization of HSP70 on genes transcribed by RNA polymerase III. HSP70 interacts with the transcribed tRNA, and this interaction alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription.
DNA double-strand breaks (DSBs) induced by abortive topoisomerase II (TOP2) activity are a potential source of genome instability and chromosome translocation. TOP2-induced DNA double-strand breaks ...are rejoined in part by tyrosyl-DNA phosphodiesterase 2 (TDP2)-dependent non-homologous end-joining (NHEJ), but whether this process suppresses or promotes TOP2-induced translocations is unclear. Here, we show that TDP2 rejoins DSBs induced during transcription-dependent TOP2 activity in breast cancer cells and at the translocation 'hotspot', MLL. Moreover, we find that TDP2 suppresses chromosome rearrangements induced by TOP2 and reduces TOP2-induced chromosome translocations that arise during gene transcription. Interestingly, however, we implicate TDP2-dependent NHEJ in the formation of a rare subclass of translocations associated previously with therapy-related leukemia and characterized by junction sequences with 4-bp of perfect homology. Collectively, these data highlight the threat posed by TOP2-induced DSBs during transcription and demonstrate the importance of TDP2-dependent non-homologous end-joining in protecting both gene transcription and genome stability.DNA double-strand breaks (DSBs) induced by topoisomerase II (TOP2) are rejoined by TDP2-dependent non-homologous end-joining (NHEJ) but whether this promotes or suppresses translocations is not clear. Here the authors show that TDP2 suppresses chromosome translocations from DSBs introduced during gene transcription.
Type II DNA topoisomerases relax topological stress by transiently gating DNA passage in a controlled cut-and-reseal mechanism that affects both DNA strands. Therefore, they are essential to overcome ...topological problems associated with DNA metabolism. Their aberrant activity results in the generation of DNA double-strand breaks, which can seriously compromise cell survival and genome integrity. Here, we profile the transcriptome of human-telomerase-immortalized retinal pigment epithelial 1 (RPE-1) cells when treated with merbarone, a drug that catalytically inhibits type II DNA topoisomerases. We performed RNA-Seq after 4 and 8 h of merbarone treatment and compared transcriptional profiles versus untreated samples. We report raw sequencing data together with lists of gene counts and differentially expressed genes.
Children with attention deficit hyperactivity disorder (ADHD) present alterations in both their attentional mechanisms and their inhibitory control processes (impulsiveness and hyperactivity), which ...have different effects on their academic, socio-emotional and behavioural achievement. Therefore it is essential to have access to intervention strategies in the field of psychopedagogics that can have a favourable effect on the developmental course of the patients.
To review the psychopedagogical foundations underlying interventions in cases of ADHD, bearing in mind the relation between the functional anatomy and the clinical symptoms and the corresponding programmes of intervention.
Three syndromes stand out above the rest: orbitofrontal, dorsolateral and in the cingulate at the medial level. The phases of psychopedagogical intervention should cover both the child and his or her family and school. Neurocognitive training is focused on the child, and is based on programmes for training the executive functions and on interventions involving the academic, behavioural and socio-affective spheres. Behavioural modification programmes are complementary and on many occasions improve the compromised behaviour in children with ADHD. Psychopedagogical guidance at school must be considered necessary for an effective intervention in the academic environment.
Psychopedagogical interventions in children with ADHD must include personalised treatment within a multidisciplinary methodology that takes into account all the contexts in which the child is developing, his or her cognitive performance and the appropriate pharmacological intervention in each case.
Topoisomerase 2 (TOP2) DNA transactions are essential for life, and proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to ...trapping by potent anticancer TOP2 drugs. How genotoxic TOP2 DNA-protein crosslinks are resolved is unclear. Here, we show that the SUMO ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent Tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a "split-SIM" SUMO2 engagement platform. These findings uncover a ZATT–TDP2 catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc.
Abstract
Background
Carbapenem-resistant Gram-negative bacilli (CR-GNB) are among the most threatening microorganisms worldwide and carbapenem use facilitates their spread. Antimicrobial stewardship ...programmes (ASPs) can help to optimize the use of antibiotics. This study evaluates the impact of a multifaceted educational ASP on carbapenem use and on the epidemiology of CR-GNB.
Methods
We conducted a quasi-experimental, time-series study in seven hospitals, from January 2014 to September 2018. The key intervention was composed of educational interviews promoting the appropriate use of carbapenems. The primary endpoints were carbapenem consumption and incidence density (ID) of CR-GNB. All non-duplicated CR-GNB clinical isolates were tested using phenotypic assays and PCR for the presence of carbapenemases. Joinpoint regression and interrupted time-series analyses were used to determine trends.
Results
A decrease in carbapenem consumption throughout the study period average quarterly percentage change (AQPC) −1.5%, P < 0.001 and a −8.170 (−16.064 to −0.277) level change following the intervention were observed. The ID of CR-Acinetobacter baumannii decreased (AQPC −3.5%, P = 0.02) and the overall ID of CR-GNB remained stable (AQPC −0.4%, P = 0.52). CR-GNB, CR-Pseudomonas aeruginosa and CR-A. baumannii IDs per hospital correlated with the local consumption of carbapenems. The most prevalent carbapenem resistance mechanisms were OXA-23 for CR-A. baumannii (76.1%), OXA-48 for CR-Klebsiella pneumoniae (66%) and no carbapenemases for CR-P. aeruginosa (91.7%). The epidemiology of carbapenemases was heterogeneous throughout the study, especially for carbapenemase-producing Enterobacteriaceae.
Conclusions
In conclusion, a multifaceted, educational interview-based ASP targeting carbapenem prescribing reduced carbapenem use and the ID of CR-A. baumannii.
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