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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Main Authors:	Broderick, Ronan, Ramadurai, Sivaramakrishnan, Tóth, Katalin, Togashi, Denisio M., Ryder, Alan G., Langowski, Jörg, Nasheuer, Heinz Peter
Format:	Online
Language:	English
Published:	Public Library of Science 2012
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334904/

id	pubmed-3334904
recordtype	oai_dc
spelling	pubmed-33349042012-04-25 Cell Cycle-Dependent Mobility of Cdc45 Determined in vivo by Fluorescence Correlation Spectroscopy Broderick, Ronan Ramadurai, Sivaramakrishnan Tóth, Katalin Togashi, Denisio M. Ryder, Alan G. Langowski, Jörg Nasheuer, Heinz Peter Research Article 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. Public Library of Science 2012-04-19 /pmc/articles/PMC3334904/ /pubmed/22536402 http://dx.doi.org/10.1371/journal.pone.0035537 Text en Broderick et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Broderick, Ronan Ramadurai, Sivaramakrishnan Tóth, Katalin Togashi, Denisio M. Ryder, Alan G. Langowski, Jörg Nasheuer, Heinz Peter
spellingShingle	Broderick, Ronan Ramadurai, Sivaramakrishnan Tóth, Katalin Togashi, Denisio M. Ryder, Alan G. Langowski, Jörg Nasheuer, Heinz Peter Cell Cycle-Dependent Mobility of Cdc45 Determined in vivo by Fluorescence Correlation Spectroscopy
author_facet	Broderick, Ronan Ramadurai, Sivaramakrishnan Tóth, Katalin Togashi, Denisio M. Ryder, Alan G. Langowski, Jörg Nasheuer, Heinz Peter
author_sort	Broderick, Ronan
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
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.
publisher	Public Library of Science
publishDate	2012
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334904/
_version_	1611524069129191424

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

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