Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz

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Braet, C; Stephan, H; Dobbie, IM; Togashi, DM; Ryder, AG; Földes-Papp, Z; Lowndes, N; Nasheuer, HP.
Mobility and distribution of replication protein A in living cells using fluorescence correlation spectroscopy.
Exp Mol Pathol. 2007; 82(2):156-162 Doi: 10.1016/j.yexmp.2006.12.008
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Führende Autor*innen der Med Uni Graz: Földes-Papp Zeno
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Abstract:: 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.
Find related publications in this database (using NLM MeSH Indexing): Cell Nucleus - metabolism; Cytosol - metabolism; DNA, Single-Stranded - metabolism; Diffusion - metabolism; Green Fluorescent Proteins - genetics; Hela Cells - genetics; Humans - genetics; Kinetics - genetics; Microscopy, Confocal - genetics; Mutation - genetics; Protein Subunits - genetics; Protein Transport - genetics; Recombinant Fusion Proteins - genetics; Replication Protein A - genetics; Spectrometry, Fluorescence - genetics
Find related publications in this database (Keywords): genome stability; DNA replication; DNA repair; replication protein A; green fluorescent protein; fluorescence correlation spectroscopy (FCS); laser scanning microscopy (LSM)