Structure of the Endonuclease Domain of MutL: Unlicensed to Cut

DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn2+-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired nicking of newly replicated DNA and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair.American Cancer Society (Research Professor)Natural Sciences and Engineering Research Council of Canada (NSERC scholarship)National Institutes of Health (U.S.) (CA21615)National Institutes of Health (U.S.) (GM45190)Natural Sciences and Engineering Research Council of Canada (NSERC, 288295)Deutsche Forschungsgemeinschaft (FR-1495/4-1)University of Michigan (Start-up funds

Learning Analytics Acceptance in Higher Education: An Extension of the TAM with Motivational and Self-Regulatory Factors

Im Zuge der digitalen Transformation der Hochschulbildung werden Learning Analytics (LA) Systeme zunehmend eingesetzt, um selbstreguliertes Lernen zu unterstützen und personalisiertes Feedback bereitzustellen. Die vorliegende Studie untersucht zentrale Einflussfaktoren auf die Akzeptanz solcher Systeme, indem das Technology Acceptance Model (TAM) um motivationale und selbstregulatorische Konstrukte erweitert wird. Darüber hinaus werden die Präferenzen der Studierenden für verschiedene Formen LA-basierter visueller Feedbackformate analysiert – mit besonderem Fokus auf Funktionen des sozialen Vergleichs. Die Ergebnisse bestätigen die Bedeutung von Benutzerfreundlichkeit und wahrgenommener Nützlichkeit und identifizieren Aufmerksamkeitskontrolle sowie Zeitmanagement als relevante Prädiktoren für die Akzeptanz von LA. Entgegen der ursprünglichen Annahme wurde sozial referenziertes Feedback als weniger interessant wahrgenommen als individualisierte Formate. Zu den Limitationen der Studie zählen das Querschnittsdesign sowie der Erhebungszeitpunkt, der vor der praktischen Nutzung des Systems lag. Dennoch liefern die Ergebnisse wichtige Erkenntnisse für eine lernendenzentrierte Gestaltung von LA-Systemen und unterstreichen die Notwendigkeit anpassbarer Feedbackstrategien, die den individuellen Präferenzen und regulatorischen Fähigkeiten der Lernenden gerecht werden.Amid the digital transformation of higher education, Learning Analytics (LA) systems are increasingly used to support self-regulated learning and provide personalized feedback. This study investigates key factors influencing students’ acceptance of such systems by extending the Technology Acceptance Model (TAM) with motivational and self-regulatory constructs. It also examines students’ preferences for different types of LA-based visual feedback, with a particular focus on social comparison features. The findings confirm the importance of usability and perceived usefulness, and identify attention control and time management as relevant predictors of LA acceptance. Contrary to initial assumptions, socially referenced feedback was perceived as less interesting than individualized formats. Limitations of the study include the cross-sectional design and the timing of data collection, which occurred before students had hands-on experience with the system. Nevertheless, the results provide important insights into learner-centered LA design and highlight the need for adaptable feedback strategies that align with individual preferences and regulatory capacities

IMPLEMENTING GENERATIVE AI IN ACADEMIC WRITING SEMINARS

Generative AI, including technologies like ChatGPT, is profoundly influencing academic writing seminars in higher education. For this reason, the use of AI tools was implemented in a case study in a structured and scientifically validated master’s seminar on academic writing. The one-semester seminar covers both business and engineering disciplines. It utilizes a defined workflow for writing an academic paper in a guided process. The new approach is that working with generative AI, e.g., ChatGPT 4.0, as an up-to-date technology is explicitly recommended, and corresponding licenses are provided to offer all participants equal conditions. Through practice and reflection students are sensitized for a productive, but also scientifically honest and responsible handling of AI tools. Data protection considerations are discussed. Practical experimentation and in-process exercises help students to assess the quality of LLM output. Surveys and observations are conducted before and during the writing process and a fina

Molecular Determinants and Consequences of Specificity in Histone 2A Ubiquitination

Specific ubiquitination of histones H2A is a crucial decision making point in the response to DNA damage. This thesis analysis the role of three distinct groups of lysines on H2A that are specifically ubiquitinated by three different E3 ligases, RING1b/BMI1, BRCA1/BARD1 and RNF168. The mechanistic basics underlying this specificity are discussed. The work describes how specific ubiquitination is employed to guide repair pathway choice between homologous recombination and non-homologous end joining. It shows that USP48, a deubiquitinating enzyme specific for the BRCA1 ubiquitination site, guides repair pathway choice by determining the extent of DNA end resection. Analysis of the E3 ligase RNF168 shows how specific interaction of the E3 with the nucleosomal acidic patch defines site-specificity. Furthermore, a general framework for structural analysis of E3-E2-Substrate complexes is presented

Histone H3.3 lysine 9 and 27 control repressive chromatin at cryptic enhancers and bivalent promoters

Histone modifications are associated with distinct transcriptional states, but it is unclear whether they instruct gene expression. To investigate this, we mutate histone H3.3 K9 and K27 residues in mouse embryonic stem cells (mESCs). Here, we find that H3.3K9 is essential for controlling specific distal intergenic regions and for proper H3K27me3 deposition at promoters. The H3.3K9A mutation resulted in decreased H3K9me3 at regions encompassing endogenous retroviruses and induced a gain of H3K27ac and nascent transcription. These changes in the chromatin environment unleash cryptic enhancers, resulting in the activation of distinctive transcriptional programs and culminating in protein expression normally restricted to specialized immune cell types. The H3.3K27A mutant disrupts the deposition and spreading of the repressive H3K27me3 mark, particularly impacting bivalent genes with higher basal levels of H3.3 at promoters. Therefore, H3.3K9 and K27 crucially orchestrate repressive chromatin states at cis-regulatory elements and bivalent promoters, respectively, and instruct proper transcription in mESCs

USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A

BRCA1 ligase activity is tightly regulated to maintain genome stability and confer DNA double strand repair. Here the authors identify USP48 as a H2A deubiquitinating enzyme that acts as a BRCA1 E3 ligase antagonist and characterize its role during DNA repair

Units for Promoter Measurement in Mammalian Cells

The purpose of this RFC is to provide units for the characterization of promoter strength for use in mammalian cells. RMPU is mRNA based and directly proportional to PoPS, whereas REU is protein based and not proportional to PoPS

A complex proinflammatory cascade mediates the activation of HSCs upon LPS exposure in vivo

Infections are a key source of stress to the hematopoietic system. While infections consume short-lived innate immune cells, their recovery depends on quiescent hematopoietic stem cells (HSCs) with long-term self-renewal capacity. Both chronic inflammatory stress and bacterial infections compromise competitive HSC capacity and cause bone marrow failure. However, our understanding of how HSCs act during acute and contained infections remains incomplete. Here, we used advanced chimeric and genetic mouse models in combination with pharmacological interventions to dissect the complex nature of the acute systemic response of HSCs to lipopolysaccharide (LPS), a well-established model for inducing inflammatory stress. Acute LPS challenge transiently induced proliferation of quiescent HSCs in vivo. This response was not only mediated via direct LPS-TLR4 conjugation on HSCs, but also involved indirect TLR4 signaling in CD115+ monocytic cells, inducing a complex pro-inflammatory cytokine cascade in the bone marrow. Downstream of LPS-TLR4 signaling, the combined action of pro-inflammatory cytokines such as IFNα, IFNγ, TNFα, IL-1α, IL-1β and many others is required to mediate full HSC activation in vivo. Together, our study reveals detailed mechanistic insights into the interplay of proinflammatory cytokine-induced molecular pathways and cell types that jointly orchestrate the complex process of emergency hematopoiesis and HSC activation upon LPS exposure in vivo

Strategy for development of site-specific ubiquitin antibodies

Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin-76 amino acids-complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments.Chemical Immunolog