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Braet, C,Stephan, H,Dobbie, IM,Togashi, DM,Ryder, AG,Foldes-Papp, Z,Lowndes, N,Nasheuer, HP
Experimental And Molecular Pathology
Mobility and distribution of replication protein A in living cells using fluorescence correlation spectroscopy
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genome stability DNA replication DNA repair replication protein A green fluorescent protein fluorescence correlation spectroscopy (FCS) laser scanning microscopy (LSM) CROSS-CORRELATION SPECTROSCOPY DNA-REPLICATION RPA PHOSPHORYLATION SINGLE-MOLECULE IN-VIVO MICROSCOPY INITIATION MEMBRANE INVITRO
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-R-PA2deltaN 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 mu m(2)/S and 1.8 mu m(2)/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. (C) 2007 Elsevier Inc. All rights reserved.
DOI 10.1016/j.yexmp.2006.12.008
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