PLoS ONE (Jan 2012)

Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy.

  • Ronan Broderick,
  • Sivaramakrishnan Ramadurai,
  • Katalin Tóth,
  • Denisio M Togashi,
  • Alan G Ryder,
  • Jörg Langowski,
  • Heinz Peter Nasheuer

DOI
https://doi.org/10.1371/journal.pone.0035537
Journal volume & issue
Vol. 7, no. 4
p. e35537

Abstract

Read online

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.