Isolierung und Identifikation von G-Quadruplex-DNA-bindenden Proteinen

  • Isolation and identification of G-Quadruplex DNA-binding proteins

Rauser, Valerie Christine; Weinhold, Elmar (Thesis advisor); Albrecht, Markus (Thesis advisor)

Aachen (2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019

Abstract

In this work, progress was made in the subject of protein isolation and identification, which bind to the biologic relevant G-quadruplex forming DNA sequence derived from the promotor of the onkogen c-myc. Development of a new methodology enabled the transformation of the polymorphic structure of the guanine-rich sequence Pu27 of the c-myc promotor to a monomeric G4 structure. Multimers were denaturized by treatment with 150 mM NaOH at room temperature. Renaturization in presence of excessive amount of K+¬-buffer lead to a selective formation of the desired parallel oriented G4-monomer. The successful application of this method could be confirmed by SEC-analyses, gel electrophoresis, CD spectroscopy and UV-melting curves for the Pu27 sequence as well as related DNA sequences. The monomerized and biotinylized G4 DNA was immobilized on streptavidin coated magnetic beads for the isolation of the G4 binding proteins from cell lysate. Beside Pu27 and its derivative Myc22, single- and double-stranded DNA was tested as control sequence. Furthermore, the displaceability of the proteins was evaluated by treatment with the G4-binding ligand PhenDC3. Analysis via SDS-PAGE showed a selective binding of many proteins to the Pu27 and Myc22 sequence, which showed no affinity for the single- and double-stranded control sequence. On the other hand, those DNA structures enabled the isolation of non-G4-binding DNA. Other proteins could be found in all samples. Many of the G4-binding proteins could be displaced by PhenDC3. For identification of the isolated proteins, the generated samples were analyzed via mass spectrometry and assigned based on a human database in collaboration with the Institute Curie in Paris, France. Analysis of the protein affinity to G4-sequence in comparison to the control sequence enabled the selection of the relevant target proteins. In a second step, the promising proteins examined regarding the displaceability with PhenDC3. Proteins with a good binding property to the examined G4-sequence as well as a good displaceability with PhenDC3 are defined as target protein. 28 target proteins meeting those criteria could be identified via this method. Next, the affinity of purified target proteins to different DNA structures was examined. Within this study, binding of identified target proteins to G4 DNA was verified and the proteins YBX1 and hnRNP U were identified as promising candidates. Alltogether, a biologic relevant, monomeric G4-structure derived from the polymorphic c-myc promotor was formed in vitro. This G4 could be utilized for the isolation of proteins from humane cancer cells. Those target proteins have been identified and their binding selectivity was validated, which enables further investigations of their biologic function.

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