CONSERVED FUNCTIONAL DOMAINS OF THE RNA-POLYMERASE-III GENERAL TRANSCRIPTION FACTOR BRF

In Saccharomyces cerevisiae, two components of the RNA polymerase III (Pol III) general transcription factor TFIIIB are the TATA-binding protein (TBP) and the B-related factor (BRF), so called because its amino-terminal half is homologous to the Pol II transcription factor IIB (TFIIB). We have cloned BRF genes from the yeasts Kluyveromyces lactis and Candida albicans, Despite the large evolutionary distance between these species and S. cerevisiae, the BRF proteins are conserved highly. Although the homology is most pronounced in the amino-terminal half, conserved regions also exist in the carboxy-terminal half that is unique to BRF. By assaying for interactions between BRF and other Pol III transcription factors, we show that it is able to bind to the 135-kD subunit of TFIIIC and also to TBP. Surprisingly, in addition to binding the TFIIB-homologous amino-terminal portion of BRF, TBP also interacts strongly with the carboxy-terminal half. Deleting two conserved regions in the BRF carboxy-terminal region abrogates this interaction. furthermore, TBP mutations that selectively inhibit Pol III transcription in vivo impair interactions between TBP and the BRF carboxy-terminal domain. Finally, we demonstrate that BRF but not TFIIB binds the Pol III subunit C34 and we define a region of C34 necessary for this interaction. These observations provide insights into the roles performed by BRF in Pol III transcription complex assembly

Dma/RNF8 proteins are evolutionarily conserved E3 ubiquitin ligases that target septins.

PublishedJournal ArticleResearch Support, Non-U.S. Gov'tThe budding yeast proteins Dma1 and Dma2 are members of the unique FHA-RING domain protein family and are linked to mitotic regulation and septin organization by ill-defined mechanisms. We show that Dma2 has ubiquitin ligase activity, and that septins Shs1 and Cdc11 are likely direct in vivo targets. We further propose that human RNF8, rather than Chfr, is the mammalian Dma homolog. As in yeast, RNF8 localizes to the centrosomes and cell division sites and promotes ubiquitylation of the septin SEPT7, whose depletion increases cell division anomalies. Together, these findings reveal evolutionary and functional conservation of Dma proteins, and suggest that RNF8 maintains genome stability through independent, yet analogous, nuclear and cytoplasmic ubiquitylation activities.HFSP fellowshipCRUKERCEuropean Community’s Seventh Framework ProgramWellcome Trus

RNF8 links nucleosomal and cytoskeletal ubiquitylation of higher order protein structures.

PublishedCommentJournal ArticleN/

Deubiquitylating enzymes and drug discovery: emerging opportunities

More than a decade after a Nobel Prize was awarded for the discovery of the ubiquitin-proteasome system and clinical approval of proteasome and ubiquitin E3 ligase inhibitors, first-generation deubiquitylating enzyme (DUB) inhibitors are now approaching clinical trials. However, although our knowledge of the physiological and pathophysiological roles of DUBs has evolved tremendously, the clinical development of selective DUB inhibitors has been challenging. In this Review, we discuss these issues and highlight recent advances in our understanding of DUB enzymology and biology as well as technological improvements that have contributed to the current interest in DUBs as therapeutic targets in diseases ranging from oncology to neurodegeneration.The authors thank L. Greger for the generation of the phylogenic tree. Research in the S.P.J. laboratory is funded by the Cancer Research UK (CRUK) Program Grant C6/A18796 and a Wellcome Trust Investigator Award (206388/Z/17/Z), and institute core infrastructure funding is provided by the CRUK (C6946/A24843) and the Wellcome Trust (WT203144)

Myocardial Fibrosis in Heart Failure: Anti-Fibrotic Therapies and the Role of Cardiovascular Magnetic Resonance in Drug Trials

All heart muscle diseases that cause chronic heart failure finally converge into one dreaded pathological process that is myocardial fibrosis. Myocardial fibrosis predicts major adverse cardiovascular events and death, yet we are still missing the targeted therapies capable of halting and/or reversing its progression. Fundamentally it is a problem of disproportionate extracellular collagen accumulation that is part of normal myocardial ageing and accentuated in certain disease states. In this article we discuss the role of cardiovascular magnetic resonance (CMR) imaging biomarkers to track fibrosis and collate results from the most promising animal and human trials of anti-fibrotic therapies to date. We underscore the ever-growing role of CMR in determining the efficacy of such drugs and encourage future trialists to turn to CMR when designing their surrogate study endpoints

ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response

In vertebrate cells, the DNA damage response is controlled by three related kinases: ATM, ATR, and DNA-PK. It has been 20 years since the cloning of ATR, the last of the three to be identified. During this time, our understanding of how these kinases regulate DNA repair and associated events has grown profoundly, although major questions remain unanswered. Here, we provide a historical perspective of their discovery and discuss their established functions in sensing and responding to genotoxic stress. We also highlight what is known regarding their structural similarities and common mechanisms of regulation, as well as emerging non-canonical roles and how our knowledge of ATM, ATR, and DNA-PK is being translated to benefit human health

Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency

The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PK$_{cs}$ and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). $\textit{In vivo}$ studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. $\textit{In vitro}$ studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5' overhangs, and 3' overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.National Institutes of Health, Cancer Research UK Program Grant IDs: C6/A11224, C6946/A14492), Wellcome Trust (Grant IDs: WT092096, WT093167

In-Situ Biofilm Formation in Hyper Alkaline Environments

Lime manufacture in the UK has resulted in the generation of a number of alkaline sites (>pH 11.0) with complex indigenous microbial populations. Within the present study, retrievable cotton samples were used to investigate the fate of cellulose, the primary carbon source, within three sites aged from ≈25 to 140 years. Following 3 months incubation in situ, biofilms had formed on all cotton samples in these extreme pH conditions; with matrices comprised of carbohydrates, proteins, lipids and eDNA. Biofilms from the older sites contained greater amounts of eDNA, a structural component that aids the production of a denser biofilm. The age of the sites correlated with a shift from polysaccharides composed of β 1,4 and β 1,3 linked sugars to those composed of pyranosyl sugars within the older sites. These changes were reflected in the active biofilm communities which shifted from being Clostridiales dominated in the youngest site to Proteobacteria dominated in the older sites. The study demonstrates that the microbial communities resident in anthropogenic alkaline sites are able to form biofilms at pH values > pH 11.0 and that these biofilms evolve toward Proteobacteria dominated communities employing eDNA and pyranosyl sugar based polysaccharides to build the biofilm matrix

E AGORA, BRECHT?

O objetivo deste artigo é analisar a intertextualidade entre a poesia de Bertold Brecht e Carlos Drummond de Andrade, sob a perspectiva da Segunda Guerra Mundial. A poesia alemã de Bertold Brecht ainda é pouco traduzida no Brasil. O autor tem vasta obra e inúmeros poemas reunidos no livro Die Gedichte von Bertold Brecht, onde retrata a dor, o sofrimento e o inconformismo declaradamente esquerdista perante o crescente nazismo na Alemanha no entre guerras. Já Drummond tem sua obra amplamente lida e apreciada. Os poemas que escolhi para esta análise são do livro A Rosa do Povo – publicado em 1945 – e considerado pela crítica como um dos mais poéticos e políticos. Busquei fundamentar a minha pesquisa em Hannah Arendt, John Willett, Erich Hobsbawm, Alfredo Bosi e Affonso Romano de Sant’Anna, entre outros.PALAVRAS CHAVE: Bertold Brecht. Carlos Drummond de Andrade. Segunda Guerra Mundial. Utopia. Abstract O objetivo deste artigo é analisar a intertextualidade entre a poesia de Bertold Brecht e Carlos Drummond de Andrade, sob a perspectiva da Segunda Guerra Mundial. A poesia alemã de Bertold Brecht ainda é pouco traduzida no Brasil. O autor tem vasta obra e inúmeros poemas reunidos no livro Die Gedichte von Bertold Brecht, onde retrata a dor, o sofrimento e o inconformismo declaradamente esquerdista perante o crescente nazismo na Alemanha no entre guerras. Já Drummond tem sua obra amplamente lida e apreciada. Os poemas que escolhi para esta análise são do livro A Rosa do Povo – publicado em 1945 – e considerado pela crítica como um dos mais poéticos e políticos. Busquei fundamentar a minha pesquisa em Hannah Arendt, John Willett, Erich Hobsbawm, Alfredo Bosi e Affonso Romano de Sant’Anna, entre outros.KEYWORDS: Bertold Brecht. Carlos Drummond de Andrade. Second World War. Utopia DOI: https://doi.org/10.47295/mren.v2i1.396