Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks.
Ku, also known as nuclear Factor IV, is an abundant nuclear DNA-binding protein which requires free DNA ends for the initial interaction with double-stranded DNA (dsDNA) and can bind at multiple sites along dsDNA in an energy-independent manner. Its function in vivo is unknown, but it has been implicated in both DNA replication and repair and in transcriptional control. We have used an electrophoretic mobility shift assay to further define the DNA binding properties of the Ku protein. Titration of Ku to a fixed amount of any of several target linear dsDNA fragments produced ladders of shifted bands proportional to the length of DNA, confirming the multiple binding activity of Ku and demonstrating its sequence-independent nature. Using a short DNA fragment with one Ku binding site, the binding constant of Ku for dsDNA ends was calculated to be 2.4 x 10(9) M-1. Competitive inhibition experiments confirmed the requirement of a free DNA end for binding by Ku and demonstrated that Ku binds isolated nicks in dsDNA. Nick binding was also observed directly using radiolabeled singly nicked circular DNA. The relative affinities of Ku for specific nick sites and free DNA ends were approximately equal, and nick binding was sequence-independent. Finally, in a study of a possible role for Ku in protecting or repairing damaged DNA, Ku was shown to inhibit the ability of T4 DNA ligase to circularize linear dsDNA molecules, demonstrating that some Ku molecules remain at the DNA terminus rather than translocate. A similar inhibition was not observed at nicks. These experiments document a new DNA binding specificity for Ku and further suggest that the high affinity end and nick binding activity is biologically relevant to its functions in vivo.
Blier
P R
PR
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Griffith
A J
AJ
Craft
J
J
Hardin
J A
JA
D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D011088:DNA Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids
D034961:Antigens, Nuclear; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D011088:DNA Ligases