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Cell Cycle-Dependent Mobility of Cdc45 Determined in vivo by Fluorescence Correlation Spectroscopy

Author

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

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.

Suggested Citation

  • Ronan Broderick & Sivaramakrishnan Ramadurai & Katalin Tóth & Denisio M Togashi & Alan G Ryder & Jörg Langowski & Heinz Peter Nasheuer, 2012. "Cell Cycle-Dependent Mobility of Cdc45 Determined in vivo by Fluorescence Correlation Spectroscopy," PLOS ONE, Public Library of Science, vol. 7(4), pages 1-10, April.
    • Handle: RePEc:plo:pone00:0035537
    DOI: 10.1371/journal.pone.0035537
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    References listed on IDEAS

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    1. Jan H. J. Hoeijmakers, 2001. "Genome maintenance mechanisms for preventing cancer," Nature, Nature, vol. 411(6835), pages 366-374, May.
    2. Nicolas Dross & Corentin Spriet & Monika Zwerger & Gabriele Müller & Waldemar Waldeck & Jörg Langowski, 2009. "Mapping eGFP Oligomer Mobility in Living Cell Nuclei," PLOS ONE, Public Library of Science, vol. 4(4), pages 1-13, April.
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