English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

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

MPS-Authors
/persons/resource/persons21520

Frank,  Joachim
Physical Chemistry, Fritz Haber Institute, Max Planck Society;

/persons/resource/persons21639

Holzwarth,  Josef F.
Physical Chemistry, Fritz Haber Institute, Max Planck Society;

External Resource
No external resources are shared
Fulltext (public)
There are no public fulltexts stored in PuRe
Supplementary Material (public)
There is no public supplementary material available
Citation

Xu, H., Frank, J., Trier, U., Hammer, S., Schröder, W., Behlke, J., et al. (2001). Interaction of fluorescence labeled single-stranded DNA with hexameric DNA-helicase RepA: A photon and fluorescence correlation spectroscopy study. Biochemistry, 40(24), 7211-7218. doi:10.1021/bi001543o.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0011-1817-1
Abstract
Fluorescence correlation spectroscopy (FCS) was used to characterize the interaction of fluorescence labeled single-stranded DMA (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 ATP gammaS at pH 5.8 and 25 degreesC were determined to be 0.58 +/- 0.12, 0.52 +/- 0.07, and 1.66 +/- 0.32 muM, 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 ATP gammaS and at pH 5.8 in the absence of ATP gammaS, 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 muM. 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 ATP<gamma>S showed a structural change induced by ssDNA binding to hRepA which is not visible at pH 7.6 and with ADP as nucleotide cofactor.