Interaction of fluorescence labeled single-stranded DNA with hexameric DNA-helicase RepA: a photon and fluorescence correlation spectroscopy study.
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, Kd(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 ATPgammaS at pH 5.8 and 25 degrees C were determined to be 0.58 +/- 0.12, 0.52 +/- 0.07, and 1.66 +/- 0.32 microM, 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 ATPgammaS and at pH 5.8 in the absence of ATPgammaS, complex formation between ssDNA and hRepA was too weak for measuring complete binding curves by FCS. Under these conditions, the dissociation constant, Kd(app), is in the range between 10 and 250 microM. 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 ATPgammaS showed a structural change induced by ssDNA binding to hRepA which is not visible at pH 7.6 and with ADP as nucleotide cofactor.
Xu
H
H
Institut für Kristallographie, Freie Universität Berlin, Takustrasse 6, D-14195 Berlin, Germany.
Frank
J
J
Trier
U
U
Hammer
S
S
Schröder
W
W
Behlke
J
J
Schäfer-Korting
M
M
Holzwarth
J F
JF
Saenger
W
W
D001665:Binding Sites; D001896:Boron Compounds / Q000378:metabolism; D002151:Calorimetry; D002942:Circular Dichroism; D004265:DNA Helicases / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli; D005456:Fluorescent Dyes / Q000378:metabolism*; D007834:Lasers; D017785:Photons; D011506:Proteins / Q000378:metabolism*; D012542:Scattering, Radiation; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods; D015534:Trans-Activators; D014461:Ultracentrifugation
C120161:BODIPY FL hydrazide; D001896:Boron Compounds; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D011506:Proteins; D012996:Solutions; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases