کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2775961 1152354 2007 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Mobility and distribution of replication protein A in living cells using fluorescence correlation spectroscopy
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوشیمی بالینی
پیش نمایش صفحه اول مقاله
Mobility and distribution of replication protein A in living cells using fluorescence correlation spectroscopy
چکیده انگلیسی

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 μm2/s and 1.8 μm2/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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Experimental and Molecular Pathology - Volume 82, Issue 2, April 2007, Pages 156–162
نویسندگان
, , , , , , , ,