TY - THES A1 - Götz, Silvia T1 - Zuo1 - ein neues G-Quadruplex-bindendes Protein in \(Saccharomyces\) \(cerevisiae\) T1 - Zuo1 - a novel G-quadruplex binding protein in \(Saccharomyces\) \(cerevisiae\) N2 - G-Quadruplex (G4)-Strukturen sind sehr stabile und polymorphe DNA und RNA Sekundärstrukturen mit einem konservierten Guanin-reichen Sequenzmotiv (G4-Motiv). Sie bestehen aus übereinander gestapelten planaren G-Quartetts, in denen je vier Guanine durch Wasserstoffbrückenbindungen zusammengehalten werden. Da G4-Motive in Eukaryoten an bestimmten Stellen im Genom angereichert vorkommen, wird angenommen, dass die Funktion von G4-Strukturen darin besteht, biologische Prozesse positiv oder negativ zu regulieren. Aufgrund der hohen thermodynamischen Stabilität von G4 Strukturen ist davon auszugehen, dass Proteine in die Faltung, Stabilisierung und Entfaltung dieser Nukleinsäure-Strukturen regulatorisch involviert sind. Bis heute wurden viele Proteine in der Literatur beschrieben, die G4-Strukturen entwinden können. Jedoch konnten bisher nur wenige Proteine identifiziert werden, die in vivo die Faltung fördern oder G4-Strukturen stabilisieren. Durch Yeast One-Hybrid (Y1H)-Screenings habe ich Zuo1 als neues G4 bindendes Protein identifiziert. In vitro Analysen bestätigten diese Interaktion und es stellte sich heraus, dass Zuo1 G4-Strukturen stabilisiert. Übereinstimmend mit den in vitro Daten konnte gezeigt werden, dass Zuo1 signifikant an G4-Motive im Genom von Saccharomyces ceresivisiae bindet. Genomweit überlappen G4-Motive, an die Zuo1 bindet, mit Stellen, an denen die DNA Replikation zum Stillstand kommt und vermehrt DNA Schäden vorkommen. Diese Ergebnisse legen nahe, dass Zuo1 eine Funktion während der DNA Reparatur oder in Zusammenhang mit dem Vorankommen der DNA Replikationsgabel hat, indem G4-Strukturen stabilisiert werden. Diese Hypothese wird außerdem durch genetische Experimente gestützt, wonach in Abwesenheit von Zuo1 die Genominstabilität zunimmt. Aufgrund dieser Daten war es möglich ein Model zu entwickeln, bei dem Zuo1 während der S-Phase G4-Strukturen bindet und stabilisiert wodurch die DNA Replikation blockiert wird. Diese Interaktion findet neben Stellen schadhafter DNA statt und unterstützt somit DNA Reparatur-Prozesse wie beispielsweise die Nukleotidexzisionsreparatur. Als weiteres potentielles G4-bindendes Protein wurde Slx9 in Y1H-Screenings identifiziert. In vitro Experimente zeigten zwar, dass Slx9 mit höherer Affinität an G4-Strukturen bindet im Vergleich zu anderen getesteten DNA Konformationen, jedoch wurde in S. cerevisiae genomweit keine signifikante Bindung an G4-Motive festgestellt. N2 - G-quadruplex (G4) structures are stable and polymorphic DNA and RNA secondary structures with a conserved Guanine-rich sequence motif (G4 motif). They consist of stacked planar G quartets that are held together by hydrogen bondings between four guanines. Because G4 motifs are enriched at specific sites in eukaryotic genomes, G4 structures are suggested to act as functional tools in the cell to regulate biological processes in a positive or negative manner. Considering the high thermodynamic stability of G4 structures it has been suggested that proteins regulate the formation, stabilization, and unfolding of this nucleic acid based structure. Up to now many proteins that unwind G4 structures have been described in the literature. But so far only a few proteins were identified that support the formation or stabilize G4 structures in vivo. Using yeast one-hybrid screenings, I identified Zuo1 as a novel G4-binding protein. In vitro studies confirmed this interaction and revealed that Zuo1 stabilizes G4 structures. In agreement with in vitro data I could show that Zuo1 binds significantly to G4 motifs in the S. cerevisiae genome. Genome-wide G4 motifs which are bound by Zuo1 overlap sites where DNA replication stalls and DNA damage is elevated. These results suggest that Zuo1 functions during the control of DNA repair or DNA replication fork progression by stabilization of G4 structures. This hypothesis is further supported by genetic assays showing that in the absence of Zuo1 genome instability is increased. On the basis of these data we propose a model in which Zuo1 binds and stabilizes G4 structures during S phase and by this block DNA replication. This interaction takes place near DNA damage sites and supports DNA repair processes such as nucleotide excision repair. Additionally, Slx9 was identified in Y1H screenings as a potential G4-binding protein. In vitro analyses showed that Slx9 interacts with higher affinity with G4 structures compared to other tested DNA conformations. However, no significant overlap with G4 motifs could be observed genome-wide in S. cerevisiae. KW - Saccharomyces cerevisiae KW - DNS-Bindungsproteine KW - DNS-Reparatur KW - DNA secondary structure KW - DNA Sekundärstruktur KW - Sekundärstruktur KW - Bäckerhefe Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-152158 ER - TY - JOUR A1 - Han, Chao A1 - Ren, Pengxuan A1 - Mamtimin, Medina A1 - Kruk, Linus A1 - Sarukhanyan, Edita A1 - Li, Chenyu A1 - Anders, Hans-Joachim A1 - Dandekar, Thomas A1 - Krueger, Irena A1 - Elvers, Margitta A1 - Goebel, Silvia A1 - Adler, Kristin A1 - Münch, Götz A1 - Gudermann, Thomas A1 - Braun, Attila A1 - Mammadova-Bach, Elmina T1 - Minimal collagen-binding epitope of glycoprotein VI in human and mouse platelets JF - Biomedicines N2 - Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin, regulating important platelet functions such as platelet adhesion and thrombus growth. Although the blockade of GPVI function is widely recognized as a potent anti-thrombotic approach, there are limited studies focused on site-specific targeting of GPVI. Using computational modeling and bioinformatics, we analyzed collagen- and CRP-binding surfaces of GPVI monomers and dimers, and compared the interacting surfaces with other mammalian GPVI isoforms. We could predict a minimal collagen-binding epitope of GPVI dimer and designed an EA-20 antibody that recognizes a linear epitope of this surface. Using platelets and whole blood samples donated from wild-type and humanized GPVI transgenic mice and also humans, our experimental results show that the EA-20 antibody inhibits platelet adhesion and aggregation in response to collagen and CRP, but not to fibrin. The EA-20 antibody also prevents thrombus formation in whole blood, on the collagen-coated surface, in arterial flow conditions. We also show that EA-20 does not influence GPVI clustering or receptor shedding. Therefore, we propose that blockade of this minimal collagen-binding epitope of GPVI with the EA-20 antibody could represent a new anti-thrombotic approach by inhibiting specific interactions between GPVI and the collagen matrix. KW - GPVI KW - collagen KW - blood platelets KW - thrombosis KW - anti-thrombotic therapies Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304148 SN - 2227-9059 VL - 11 IS - 2 ER -