Cell cycle-dependent mobility of Cdc45 in living cells determined by fluorescence correlation spectroscopy.
Togashi, Denisio M.
Ryder, Alan G.
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R. Broderick, S. Ramadurai, K. Toth, D. Togashi, A. G. Ryder, J. Langwoski, and H.P. Nasheuer. (2012) 'Cell cycle-dependent mobility of Cdc45 in living cells determined by fluorescence correlation spectroscopy'. Plos One, 7 (4):e35537-e35537.
Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. We measured the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS) in vivo in asynchronous cells and in cells synchronized at the G1/S transition and during S phase. Our data show that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the in vivo FCS data. Analysis of the mobility of eGFP-Cdc45 and the size ofcomplexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. This suggests that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damage.