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Interaction of fluorescence labeled single-stranded DNA with hexameric DNA-helicase RepA: A photon and fluorescence correlation spectroscopy study

Item Type:Article
Title:Interaction of fluorescence labeled single-stranded DNA with hexameric DNA-helicase RepA: A photon and fluorescence correlation spectroscopy study
Creators Name:Xu, H. and Frank, J. and Trier, U. and Hammer, S. and Schroeder, W. and Behlke, J. and Schaefer-Korting, M. and Holzwarth, J.F. and Saenger, W.
Abstract:Fluorescence correlation spectroscopy (FCS) was used to characterize the interaction of fluorescence labeled single-stranded DNA (ssDNA) with hexameric RepA DNA-helicase (hRepA) encoded by plasmid RSF1010. The apparent dissociation constants, K d(app), for the equilibrium binding of 12mer, 30mer, and 45mer ssDNA 5′-labeled with BFL to hRepA dimer in the presence of 0.5 mM ATPγS at pH 5.8 and 25 °C were determined to be 0.58 ± 0.12, 0.52 ± 0.07, and 1.66 ± 0.32 μM, respectively. Binding curves are compatible with one binding site for ssDNA present on hRepA dimer, with no indication of cooperativity. At pH 7.6 in the presence of ATPγS and at pH 5.8 in the absence of ATPγS, complex formation between ssDNA and hRepA was too weak for measuring complete binding curves by FCS. Under these conditions, the dissociation constant, K d(app), is in the range between 10 and 250 μM. The kinetics of complex formation at pH 5.8 are faster than the time resolution (approximately 10-20 s) of FCS experiments under pseudo-first-order conditions, with respect to BFL-ssDNA. Photon correlation spectroscopy (PCS) experiments yielded, within the experimental error range, the same values for the apparent hydrodynamic radii, R h, of hRepA dimer and its complex with ssDNA as determined by FCS (R h = 6.6 ± 1 nm). hRepA starts to aggregate under acidic conditions (<pH 6.0) which are optimal for ssDNA binding. CD spectra taken at pH 5.8 in the presence of ATPγS showed a structural change induced by ssDNA binding to hRepA which is not visible at pH 7.6 and with ADP as nucleotide cofactor.
Keywords:Bacterial DNA, Binding Sites, Boron Compounds, Calorimetry, Circular Dichroism, Dimerization, DNA-Binding Proteins, DNA Helicases, Escherichia Coli, Fluorescent Dyes, Fluorescence Spectrometry, Lasers, Photons, Proteins, Radiation Scattering, Single-Stranded DNA, Solutions, Trans-Activators, Ultracentrifugation
Source:Biochemistry
ISSN:0006-2960
Publisher:American Chemical Society (U.S.A.)
Volume:40
Number:24
Page Range:7211-7218
Date:1 January 2001
Official Publication:https://doi.org/10.1021/bi001543o
PubMed:View item in PubMed

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