Quantitative Fluorescence Microscopy of Protein Dynamics in Living Cells

The advent of confocal microscopy, fast microcomputers with high storage capacity and, moreover, the availability of fluorescent proteins of various excitation and emission properties have made fluorescence microscopy the method of choice in the study of protein behaviour in living cells. In this thesis we investigated in detail two important quantitative methods, fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). Model systems used in demonstrating the complementarity of the techniques and their merits included the nuclear excision repair (NER) system, transcription regulation by the androgen receptor (AR), and signal transduction by two membrane receptors, the EGF receptor and the IL2-receptor. In Chapter 1 an introduction to microscopy is given. A brief history traces the development of microscopy from the modest lens arrangement of Zacharias Janssen to modern fluorescence microscopes allowing quantitative investigation of protein dynamics in living cells. A discussion of fluorescence properties of the GFP is presented and several quantitative fluorescence microscopy techniques used are discussed. Also the model systems studied are described. In Chapter 2 the long-lived dark state of EGFP, the fluorescent tag used in most live cell studies, is investigated as observed in a set-up similar to a typical fluorescence recovery after photobleaching (FRAP) experiment. A method is presented to measure light in

Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced DNA damage depends on functional TFIIH

The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process. Copyrigh