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MOBILITY OF GFP-XPC FUSION PROTEIN IN LIVING CELL NUCLEUS OBSERVED BY TIME-LAPSE FLUORESCENCE MICROSCOPY AND FLUORESCENCE CORRELATION SPECTROSCOPY.  H. Okumura¹, A. Tanaka^1,2, Y. Kodama³, F. Ishidate³, K. Sugasawa^4,5, and F. Hanaoka^4,5,6.  ¹NASDA, ²GSC, RIKEN, ³Carl Zeiss, ⁴RIKEN and ⁵CREST, ⁶Osaka Univ Japan.

     Nucleotide excision repair (NER) is a principal pathway by which a large variety of DNA damage is eliminated from the genome. We have interest in how does the NER system monitor the lesions in huge genome DNA molecules. To study nuclear localization and dynamics of a DNA damage-binding factor XPC-hHR23B, the initiator of global genome NER, we observed time-lapse fluorescence microscopy and fluorescence correlation spectroscopy (FCS) of the GFP-XPC fusion protein in living cells.

     Localization and mobility of the GFP-XPC fusion protein were studied in XPC-deficient human primary fibroblasts (XP4KA) that had been transfected with a plasmid transiently expressing the fusion protein.  In most transfected cells, GFP-XPC was observed within nucleus, whereas control GFP localized both in nucleus and cytoplasm. We found two sorts of fluorescent components, small particles and larger speckles, in the GFP-XPC transfected cells. Following by time-lapse microscopy, the small particles moved quickly and the speckles moved slowly.

     Using FCS, we have determined diffusion coefficients of the fusion protein in living cells. Although the obtained values exhibited insufficient reproducibility, automated analysis suggested the presence of two types of fluorescent components in the nucleus. One had a diffusion time corresponding to the molecular weight of the fusion protein, and while the other had a 100 times larger diffusion time. These results suggest that in living cell nucleus GFP-XPC may behave in a large cluster which is susceptible to be effected by gravity.