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

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

doi:10.1371/journal.pone.0035537

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.

Highlights

Duplication of chromosomal DNA is an essential process for both normal cell division and to maintain stability of the genome [1]
This allows the binding of additional proteins such as Cdc6 and Cdt1 (Cdc10-dependent target) to origin recognition complex (ORC) mediating the loading of the Mcm2–7 complex to chromatin, forming the pre-replicative complex [8,9]
This activation of the helicase function of Mcm2–7 allows the formation of a larger multi-subunit protein machinery required for the elongation phase of DNA replication [10,11] and of single-stranded DNA, which is coated by RPA

Summary

Introduction

Duplication of chromosomal DNA is an essential process for both normal cell division and to maintain stability of the genome [1]. Accurate duplication of the genome is carried out by the ‘‘replisome progression complex’’ (RPC), a large multi-subunit complex consisting of replication proteins These proteins work in concert at different stages of the cell cycle to facilitate DNA replication [2,3,4,5,6,7]. Activation of the preRC is mediated by CDKs (cyclin-dependent kinases) and DDK (Dbf4-dependent kinase) to allow the binding of Cdc and the GINS (go-ichi-ni-san (five-onetwo-three)) complex to the Mcm2–7 [8,9,10] This activation of the helicase function of Mcm allows the formation of a larger multi-subunit protein machinery required for the elongation phase of DNA replication [10,11] and of single-stranded DNA, which is coated by RPA (replication protein A). RFC and PCNA, together with RPA, allow a polymerase switch from Pol-prim to Pol (DNA polymerase) e or d, synthesizing the bulk DNA synthesis on the leading strand and lagging strand, respectively [8,9]

Methods

Results

Conclusion