Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks.

摘要: Ku, also known as nuclear Factor IV, is an abundant DNA-binding protein which requires free DNA ends for the initial interaction with double-stranded (dsDNA) and can bind at multiple sites along dsDNA in energy-independent manner. Its function vivo unknown, but it has been implicated both replication repair transcriptional control. We have used electrophoretic mobility shift assay to further define binding properties of Ku protein. Titration a fixed amount any several target linear fragments produced ladders shifted bands proportional length DNA, confirming activity demonstrating its sequence-independent nature. Using short fragment one site, constant was calculated be 2.4 x 10(9) M-1. Competitive inhibition experiments confirmed requirement end by demonstrated that binds isolated nicks dsDNA. Nick observed directly using radiolabeled singly nicked circular DNA. The relative affinities specific nick were approximately equal, sequence-independent. Finally, study possible role protecting or repairing damaged shown inhibit ability T4 ligase circularize molecules, some molecules remain terminus rather than translocate. A similar not nicks. These document new specificity suggest high affinity biologically relevant functions vivo.