Studies on the mode of Ku interaction with DNA.
The Ku heterodimer plays a central role in non-homologous end-joining. The binding of recombinant Ku to DNA has been investigated by dynamic light scattering, double-filter binding, fluorescence spectroscopy, and band shift assays. The hydrodynamic radius of Ku in solution is 5.2 nm and does not change when a 25-bp double-strand DNA (dsDNA) fragment (D25) is added, indicating that only one Ku molecule binds to a 25-bp fragment. The dissociation constant (k(d)) for the binding to D25 is 3.8 +/- 0.9 nm. If both ends of the substrate are closed with hairpin loops, Ku is still able to bind with little change in the k(d). The k(d) is not affected by ATP, Mg(2+), or ionic strength. However, the addition of bovine serum albumin decreases the k(d) by 2-fold. DNA substrates of 50 bp can bind two Ku molecules, whereas three molecules are bound to a 75-bp substrate. Data analysis with the Hill equation yields a value of the Hill coefficient (n) close to 1, and the k(d) values for the binding of Ku to both ends of these substrates are the same. Thus, we demonstrate that there is no cooperative interaction among the Ku heterodimers binding longer substrates.
Arosio
Daniele
D
International Centre for Genetic Engineering and Biotechnology, Padriciano, 99, Trieste I-34012, Italy.
Cui
Sheng
S
Ortega
Claudia
C
Chovanec
Miroslav
M
Di Marco
Stefania
S
Baldini
Giancarlo
G
Falaschi
Arturo
A
Vindigni
Alessandro
A
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D034961:Antigens, Nuclear; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D007477:Ions; D007700:Kinetics; D000072200:Ku Autoantigen; D008027:Light; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D012709:Serum Albumin / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013997:Time Factors
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D007477:Ions; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D008274:Magnesium