Quantitative fluorescence nanospectroscopy of nucleotide excision repair - from single molecules to cells
Promotion date: 11 December 2008
Thesis advisor: Prof. Vinod Subramaniam
Assistant thesis advisor: Dr. Cees Otto
The work described in this thesis is for the project: “In vitro en in vivo studies of the architecture of nucleotide excision repair complexes”, granted by the Human Frontiers in Science Program. The goal was the study of damage recognition, complex assembly and mechanism of human NER, using designed photoreactive and fluorescent NER substrates.
The object is to develop and use new instrumentation and methods for the quantitative fluorescent nanospectroscopic techniques.
Finally development and use took place of: single-molecule fluorescence confocal microscopy (SFCM) and total internal reflection fluorescence (TIRF) microscopy.
Can you describe your expertise, using microscopic techniques in this thesis project?
Basically our SFCM microscope has developed in our group for about ten years. Now starting from my PhD project, the SFCM has been extended with many functions. Most experimental measurements and data processing can be performed on-line. We demonstrated all functions in the multiparameter fluorescence spectroscopic study on single living XP4PA cells.
With the help of these techniques and also using fluorescence microscopy, we studied protein stability and binding reactions in NER proteins and DNA substrates.
In the beginning of the project, what was your basic concern?
We encountered many difficulties as to choose the methods to study. There existed a lot of differences between the methods. It took some time to solve these problems first.
Did you use the MESA+ cleanroom a lot?
The investigations were carried out in many physical and chemical laboratories all over the world. The cleanroom was not necessary for our measurements. In the future I hope to use the microscopic techniques, present at MESA+, quite a lot. I am promised so already. MESA+ owns very advanced techniques and knowledge in this field, which are internationally recognized.
What are your future plans?
First of all, I have to learn quite a lot still. I’m hoping to give the research a follow-up at Erasmus medical centre, Rotterdam, studying different aspects of DNA-repair – double strand break repair.
What’s the main conclusion, looking back on this thesis project?
The developed quantitative nanospectroscopic techniques presented in this thesis, are excellent to determine the kinetics and dynamics of DNA-protein interactions, and the assembly-order of NER-proteins on DNA substrates in NER complexes, as well as the composition of the NER complex at various stages of the NER-process. The TIRF microscopy is most useful for determining the precise location of different proteins in NER complexes. All these techniques provide a bright future for the study of the architecture of NER machinery, as well as other biological systems of interest.