Mechanistic studies of protein-DNA interactions by single molecule atomic force microscopy

Mechanistische Untersuchungen von protein-DNA-Wechselwirkungen mittels Einzelmolekül-Rasterkraftmikroskopie

Please always quote using this URN: urn:nbn:de:bvb:20-opus-252047
  • Protein-DNA interactions are central to many biological processes and form the bedrock of gene transcription, DNA replication, and DNA repair processes. Many proteins recognize specific sequences in DNA- a restriction enzyme must only cut at the correct sequence and a transcription factor should bind at its consensus sequence. Some proteins are designed to bind to specific structural or chemical features in DNA, such as DNA repair proteins and some DNA modifying enzymes. Target-specific DNA binding proteins initially bind to non-specific DNAProtein-DNA interactions are central to many biological processes and form the bedrock of gene transcription, DNA replication, and DNA repair processes. Many proteins recognize specific sequences in DNA- a restriction enzyme must only cut at the correct sequence and a transcription factor should bind at its consensus sequence. Some proteins are designed to bind to specific structural or chemical features in DNA, such as DNA repair proteins and some DNA modifying enzymes. Target-specific DNA binding proteins initially bind to non-specific DNA and then search for their target sites through different types of diffusion mechanisms. Atomic force microscopy (AFM) is a single-molecule technique that is specifically well-suited to resolve the distinct states of target-specific as well as nonspecific protein-DNA interactions that are vital for a deeper insight into the target site search mechanisms of these enzymes. In this thesis, protein systems involved in epigenetic regulation, base excision repair (BER), and transcription are investigated by single-molecule AFM analyses complemented by biochemical and biophysical experiments. The first chapter of this thesis narrates the establishment of a novel, user-unbiased MatLab-based tool for automated DNA bend angle measurements on AFM data. This tool has then been employed to study the initial lesion detection step of several DNA glycosylases. These results promoted a model describing the altered plasticities of DNA at the target lesions of DNA glycosylases as the fundamental mechanism for their enhanced efficiency of lesion detection. In the second chapter of this thesis, the novel automated tool has been further extended to provide protein binding positions on the DNA along with corresponding DNA bend angles and applied to the study of DNMT3A DNA methyltransferase. These AFM studies revealed preferential co-methylation at specific, defined distances between two CpG sites by the enzyme and when combined with biochemical analyses and structural modelling supported novel modes of CpG co-methylation by DNMT3A. In the third chapter of this thesis, the role of 8-oxo-guanine glycosylase (hOGG1) in Myc-mediated transcription initiation has been investigated. AFM analyses revealed that in the presence of oxidative damage in DNA, Myc is recruited to its target site (E-box) by hOGG1 through direct protein-protein interactions, specifically under oxidizing conditions. Intriguingly, oxidation of hOGG1 was further observed to result in dimerization of hOGG1, which may also play a role in the mechanism of transcription regulation by hOGG1 under oxidative stress.show moreshow less
  • Protein-DNA-Wechselwirkungen sind für viele biologische Prozesse von zentraler Bedeutung und bilden die Grundlage der Gentranskription, der DNA-Replikation und der DNA-Reparaturprozesse. Viele Proteine erkennen bestimmte Bassen-Sequenzen in der DNA - ein Restriktionsenzym darf nur an der richtigen Sequenz schneiden, und ein Transkriptionsfaktor sollte an seine Konsenssequenz binden. Einige Proteine sind darauf ausgelegt, an bestimmte strukturelle oder chemische Merkmale der DNA zu binden, wie z. B. DNA-Reparaturproteine und verschiedeneProtein-DNA-Wechselwirkungen sind für viele biologische Prozesse von zentraler Bedeutung und bilden die Grundlage der Gentranskription, der DNA-Replikation und der DNA-Reparaturprozesse. Viele Proteine erkennen bestimmte Bassen-Sequenzen in der DNA - ein Restriktionsenzym darf nur an der richtigen Sequenz schneiden, und ein Transkriptionsfaktor sollte an seine Konsenssequenz binden. Einige Proteine sind darauf ausgelegt, an bestimmte strukturelle oder chemische Merkmale der DNA zu binden, wie z. B. DNA-Reparaturproteine und verschiedene DNA-modifizierende Enzyme. Zielspezifische DNA-bindende Proteine binden zunächst an unspezifische DNA und suchen dann durch verschiedene Arten von Diffusionsmechanismen nach ihren Zielstellen in der DNA. AFM ist eine Einzelmolekültechnik, die besonders gut geeignet ist, um die verschiedenen Zustände sowohl der spezifisch gebundenen als auch unspezifischen Protein-DNA-Wechselwirkungen aufzulösen, die für einen tieferen Einblick in die Mechanismen der Zielstellensuche unerlässlich sind. In dieser Arbeit werden Proteinsysteme, die an der epigenetischen Regulation, der Basenexzisionsreparatur (BER) und der Transkription beteiligt sind, durch Einzelmolekül- AFM-Analysen untersucht, und diese Studien werden durch biochemische und biophysikalische Experimente komplementiert. ...show moreshow less

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Metadaten
Author: Disha Mohan BangaloreGND
URN:urn:nbn:de:bvb:20-opus-252047
Document Type:Doctoral Thesis
Granting Institution:Universität Würzburg, Graduate Schools
Faculties:Graduate Schools / Graduate School of Life Sciences
Fakultät für Biologie / Rudolf-Virchow-Zentrum
Referee:PD Dr. Ingrid Tessmer
Date of final exam:2021/12/21
Language:English
Year of Completion:2022
DOI:https://doi.org/10.25972/OPUS-25204
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
GND Keyword:Transcription; atomic force microscopy
Tag:protein-DNA interactions
Release Date:2022/12/23
Licence (German):License LogoCC BY-NC: Creative-Commons-Lizenz: Namensnennung, Nicht kommerziell 4.0 International