Role of the Srs2-Rad51 Interaction Domain in Crossover Control in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Srs2, in addition to its well-documented antirecombination activity, has been proposed to play a role in promoting synthesis-dependent strand annealing (SDSA). Here we report the identification and characterization of an SRS2 mutant with a single amino acid substitution (srs2-F891A) that specifically affects the Srs2 pro-SDSA function. This residue is located within the Srs2-Rad51 interaction domain and embedded within a protein sequence resembling a BRC repeat motif. The srs2-F891A mutation leads to a complete loss of interaction with Rad51 as measured through yeast two-hybrid analysis and a partial loss of interaction as determined through protein pull-down assays with purified Srs2, Srs2-F891A, and Rad51 proteins. Even though previous work has shown that internal deletions of the Srs2-Rad51 interaction domain block Srs2 antirecombination activity in vitro, the Srs2-F891A mutant protein, despite its weakened interaction with Rad51, exhibits no measurable defect in antirecombination activity in vitro or in vivo Surprisingly, srs2-F891A shows a robust shift from noncrossover to crossover repair products in a plasmid-based gap repair assay, but not in an ectopic physical recombination assay. Our findings suggest that the Srs2 C-terminal Rad51 interaction domain is more complex than previously thought, containing multiple interaction sites with unique effects on Srs2 activity

Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops.

Synthesis-dependent strand annealing (SDSA) is the preferred mode of homologous recombination in somatic cells leading to an obligatory non-crossover outcome, thus avoiding the potential for chromosomal rearrangements and loss of heterozygosity. Genetic analysis identified the Srs2 helicase as a prime candidate to promote SDSA. Here, we demonstrate that Srs2 disrupts D-loops in an ATP-dependent fashion and with a distinct polarity. Specifically, we partly reconstitute the SDSA pathway using Rad51, Rad54, RPA, RFC, DNA Polymerase δ with different forms of PCNA. Consistent with genetic data showing the requirement for SUMO and PCNA binding for the SDSA role of Srs2, Srs2 displays a slight but significant preference to disrupt extending D-loops over unextended D-loops when SUMOylated PCNA is present, compared to unmodified PCNA or monoubiquitinated PCNA. Our data establish a biochemical mechanism for the role of Srs2 in crossover suppression by promoting SDSA through disruption of extended D-loops

Analytical techniques for multiplex analysis of protein biomarkers

Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine

International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist

Studies on functions of the genes MPH1 and MMS2 from Saccharomyces cerevisiae during error free bypass of replication blocking DNA-lesions

Die Umgehung von replikationsarretierenden DNA-Schäden läuft in vivo mindestens über zwei Prozesse: homologe Rekombination durch Schwesterchromatid-Interaktion (SCI) und Postreplikative Reparatur (PRR). Die PRR wird zusätzlich in einen fehlerfreien und einen fehlerbehafteten Weg unterteilt. Für homologe Rekombination sind vor allem die Proteine Rad51 und Rad52 notwendig. Die fehlerbehaftete PRR erfolgt über Rev3- und Rad30-abhängige Transläsionssynthese. Der Mechanismus der fehlerfreien PRR ist dagegen noch weitgehend unklar. Bekannt ist jedoch, dass die fehlerfreien Prozesse über Polyubiquitinierung des PCNA am Lysin 164 durch das Heterodimer Mms2/Ubc13 und Rad5 stimuliert werden. Die Polyubiquitinierung erfolgt über das Ubiquitin-interne Lysin 63. Nach bisheriger etablierter Lehrmeinung gelten die fehlerfreie Postreplikative Reparatur und homologe Rekombination als zwei voneinander unabhängige und alternativ einsetzbare Prozesse. Die in dieser Arbeit gewonnenen Daten zeigen dagegen, dass es sich bei der fehlerfreien PRR nicht um HR-unabhängige Prozesse handeln kann: o rad51 ist epistatisch zu mms2 in Bezug auf die Sensitivität gegen 4 NQO. o rad51 ist epistatisch zu mms2 in Bezug auf die spontanen Mutationsraten. o Wie in rad51-Mutanten ist die Rate spontaner Schwesterchromatid-Interaktionen in mms2-Mutanten ist leicht erhöht. o Die Frequenz 4 NQO-induzierter Schwesterchromatid-Interaktionen ist in mms2- sowie in ubc13-Mutanten stark reduziert. o Der ubc13-Phänotyp ist komplementierbar und von der katalytischen Funktion des Ubc13-Proteins abhängig. o Die Mutation des Ubiquitin-internen Lysin 63 führt zu einem mit ubc13 vergleichbaren Phänotyp. o Die Mutation des Substrats (PCNA-K164R) führt zu einem ähnlichen Phänotyp wie in mms2 und ubc13 und ist epistatisch zu ubc13 in Bezug auf die Sensitivität gegen 4 NQO. Die Schwesterchromatid-Interaktionen wurden im Rahmen dieser Arbeit mit einem System gemessen, welches neu in der Arbeitsgruppe von Dr. W. Kramer entwickelt wurde. Es beruht auf der Messung einer G418-Resistenz als Folge der Reversion einer direkten Sequenzwiederholung. Anhand der in dieser Arbeit dargelegten Daten lässt sich zeigen, dass die mit diesem System gemessenen Ereignisse strikt von der homologen Rekombination abhängig sind: o rad52-Mutanten weisen weder spontane noch 4-NQO-induzierte Reversions-ereignisse auf. o rad51-Mutanten weisen stark reduzierte 4 NQO-induzierte Reversionsfrequenzen auf. o Die spontane Reversionsrate in rad51 ist erhöht, was vermutlich auf eine Anhäufung von SSA- gegenüber SDSA- bzw. DSBR-Ereignissen zurückzuführen ist. Durch genetische Interaktionsstudien zwischen rad30, rev3, rad51 und mms2 konnten Evidenzen gefunden werden, welche es wahrscheinlich machen, dass die Polyubiquitinierung auch Rev3-abhängige Mechanismen stimuliert und einen Einfluss auf zusätzliche Prozesse hat: o rad51 und rev3 sind synergistisch bei der Sensitivität gegen MMS. Die zusätzliche Deletion von mms2 hat jedoch keinen weiteren Einfluss auf die Sensitivität. o rad51rad30 und mms2 sind additiv bis synergistisch bei der Sensitivität gegen UV-Strahlung. mms2 und rad51rev3 sind dagegen nur subadditiv. Für die Funktion von Mms2/Ubc13 wurden anhand dieser Daten als wahrscheinlichstes Modell eine Stimulation der Rev3-abhängigen Prozesse sowie der homologen Rekombination durch direkte Interaktion zwischen ubiquitiniertem PCNA und den beteiligten Proteinen vorgeschlagen. Für Mph1, welches an der HR-abhängigen fehlerfreie Umgehung von replikationsarre-tierenden DNA-Schäden beteiligt ist, konnten die in dieser Arbeit gewonnenen Daten zeigen, das die kürzlich in der Literatur beschriebene Auflösung eines D-Loops nicht die einzige Funktion des Proteins sein kann: o mph1 weist eine Reduktion der Camptothecin-, MMS- und 4-NQO-induzierten Reversionsfrequenzen auf, wobei die Reduktion bei 4-NQO am deutlichsten ist. o mph1 weist eine zu rad51 vergleichbare Erhöhung der spontanen Reversionsrate auf. o mph1 weist kaum eine Reduktion der 4-NQO-induzierten Reversionsfrequenzen im his3-5"/his3-3"-Modul (Fasullo & Davis, 1987, Proc Natl Acad Sci USA, 84, 6215-6219) auf, welches vermutlich hauptsächlich cross over-Ereignisse detektiert

Modularly Constructed Synthetic Granzyme B Molecule Enables Interrogation of Intracellular Proteases for Targeted Cytotoxicity

Targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. However, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. In contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. Here, we report a protein-based therapeutic platform-termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT)-which enables the interrogation of intracellular proteases to trigger targeted cytotoxicity. COVERT molecules consist of the cytotoxic protein granzyme B (GrB) fused to an inhibitory N-terminal peptide, which can be removed by researcher-specified proteases to activate GrB function. We demonstrate that fusion of a small ubiquitin-like modifier 1 (SUMO1) protein to GrB yields a SUMO-GrB molecule that is specifically activated by the cancer-associated sentrin-specific protease 1 (SENP1). SUMO-GrB selectively triggers apoptotic phenotypes in HEK293T cells that overexpress SENP1, and it is highly sensitive to different SENP1 levels across cell lines. We further demonstrate the rational design of additional COVERT molecules responsive to enterokinase (EK) and tobacco etch virus protease (TEVp), highlighting the COVERT platform's modularity and adaptability to diverse protease targets. As an initial step toward engineering COVERT-T cells for adoptive T-cell therapy, we verified that primary human T cells can express, package, traffic, and deliver engineered GrB molecules in response to antigen stimulation. Our findings set the foundation for future intracellular-antigen-responsive therapeutics that can complement surface-targeted therapies

Four hurdles for conservation on private land: the case of the golden lion tamarin, Atlantic Forest, Brazil.

Many threatened species worldwide rely on patches of remnant vegetation in private landholdings. To establish private reserves that contribute effectively to conservation involves a wide range of complex and interacting ecological, legal, social and financial factors. These can be seen as a series of successive hurdles, each with multiple bars, which must all be surmounted. The golden lion tamarin, Leontopithecus rosalia, is restricted to the Atlantic Forest biome in the state of Rio de Janeiro, Brazil. This forest is largely cleared. There are many small remnant patches on private lands, able to support tamarins. Local NGO’s have successfully used limited funds to contribute to tamarin conservation in a highly cost effective way. We examined the mechanisms by analysing documents and interviewing landholders and other stakeholders. We found that the local NGOs successfully identified landholdings where ecological, legal, social and some financial hurdles had already been crossed, and helped landholders over the final financial hurdle by funding critical cost components. This cost <5% of the price of outright land purchase. This approach is scaleable for golden lion tamarin elsewhere within the Atlantic Forest biome, and applicable for other species and ecosystems worldwide

Overlapping multivoxel patterns for two levels of visual expectation

According to predictive accounts of perception, visual cortical regions encode sensory expectations about the external world, and the violation of those expectations by inputs (surprise). Here, using multi-voxel pattern analysis (MVPA) of functional magnetic resonance imaging (fMRI) data, we asked whether expectations and surprise activate the same pattern of voxels, in face-sensitive regions of the extra-striate visual cortex (the fusiform face area or FFA). Participants viewed pairs of repeating or alternating faces, with high or low probability of repetitions. As in previous studies, we found that repetition suppression (the attenuated BOLD response to repeated stimuli) in the FFA was more pronounced for probable repetitions, consistent with it reflecting reduced surprise to anticipated inputs. Secondly, we observed that positive- and negative-going responses to repetition were both consistent across scanner runs, suggesting that both have functional significance, with repetition enhancement possibly indicating the build up of sensory expectation. Critically, we also report that multi-voxels patterns associated with probability and repetition effects were significantly correlated within the left FFA. We argue that repetition enhancement responses and repetition probability effects can be seen as two types of expectation signals, occurring simultaneously, although at different levels (lower vs. higher), and different time scales (immediate vs. long term)