Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy.
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 da...
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doaj-047d8be4478e45f3a39ad306bcde3cf12020-11-25T02:08:51ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0174e3553710.1371/journal.pone.0035537Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy.Ronan BroderickSivaramakrishnan RamaduraiKatalin TóthDenisio M TogashiAlan G RyderJörg LangowskiHeinz Peter NasheuerEukaryotic 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.http://europepmc.org/articles/PMC3334904?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Ronan Broderick Sivaramakrishnan Ramadurai Katalin Tóth Denisio M Togashi Alan G Ryder Jörg Langowski Heinz Peter Nasheuer |
spellingShingle |
Ronan Broderick Sivaramakrishnan Ramadurai Katalin Tóth Denisio M Togashi Alan G Ryder Jörg Langowski Heinz Peter Nasheuer Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. PLoS ONE |
author_facet |
Ronan Broderick Sivaramakrishnan Ramadurai Katalin Tóth Denisio M Togashi Alan G Ryder Jörg Langowski Heinz Peter Nasheuer |
author_sort |
Ronan Broderick |
title |
Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. |
title_short |
Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. |
title_full |
Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. |
title_fullStr |
Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. |
title_full_unstemmed |
Cell cycle-dependent mobility of Cdc45 determined in vivo by fluorescence correlation spectroscopy. |
title_sort |
cell cycle-dependent mobility of cdc45 determined in vivo by fluorescence correlation spectroscopy. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2012-01-01 |
description |
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. |
url |
http://europepmc.org/articles/PMC3334904?pdf=render |
work_keys_str_mv |
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