Mobility and distribution of replication protein A in living cells using fluorescence correlation spectroscopy.
Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for all pathways of DNA metabolism. To study the function of RPA in living cells the second largest RPA subunit and an N-terminal deletion mutant thereof were fused to green fluorescent protein (GFP; GFP-RPA2 and GFP-RPA2deltaN, respectively) in a controlled, molecular biological way. These proteins were expressed in HeLa cells under the control of the inducible tetracycline expression system. GFP-RPA2 and GFP-RPA2deltaN are predominately nuclear proteins as determined by confocal laser scanning microscopy. Low basal expression of GFP-RPA2deltaN allowed the measurement of kinetic parameters of RPA. Using fluorescence correlation spectroscopy (FCS) two populations--a fast and a slow moving species--were detected in the nucleus and the cytosol of human cells. The translational diffusion rates of these two RPA populations were approximately 15 microm2/s and 1.8 microm2/s. This new finding reveals the existence of different multiprotein and ssDNA-protein complexes of RPA in both cellular compartments and opens the possibility for their analyses.
Braet
Christophe
C
National University of Ireland, Galway, Department of Biochemistry, Cell Cycle Control Laboratory, Galway, Ireland.
Stephan
Holger
H
Dobbie
Ian M
IM
Togashi
Denisio M
DM
Ryder
Alan G
AG
Földes-Papp
Zeno
Z
Lowndes
Noel
N
Nasheuer
Heinz Peter
HP
D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D009154:Mutation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; D049452:Green Fluorescent Proteins; C516794:RPA2 protein, human