title: Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy.
abstract: 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 of complexes 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.
authors.affiliation: Systems Biology Ireland and Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Ronan
authors.initials: R
authors.lastname: Broderick
authors.forename: Sivaramakrishnan
authors.initials: S
authors.lastname: Ramadurai
authors.forename: Katalin
authors.initials: K
authors.lastname: Tóth
authors.forename: Denisio M
authors.initials: DM
authors.lastname: Togashi
authors.forename: Alan G
authors.initials: AG
authors.lastname: Ryder
authors.forename: Jörg
authors.initials: J
authors.lastname: Langowski
authors.forename: Heinz Peter
authors.initials: HP
authors.lastname: Nasheuer
mesh_terms: D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism / Q000528:radiation effects; D002850:Chromatography, Gel; D004249:DNA Damage; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays
chemical_list: C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins