DNA length-dependent cooperative interactions in the binding of Ku to DNA

Despite its central role in the nonhomologous DNA end joining process, we s till have an incomplete picture of the interaction between Ku and DNA. Here we describe both kinetic (surface plasmon resonance or SPR) and equilibriu m (electrophoretic mobility shift assay or EMSA) studies of Ku binding to l inear double-stranded DNA. Ku interaction with I-site DNA is noncooperative , as expected. Electrophoretic mobility shift assays indicate cooperativity in the binding of Ku molecules to DNA long enough for two Ku molecules to bind (2-site DNA). For the kinetic studies, we use surface plasmon resonanc e in which one end of the DNA molecules is linked to a surface while the ot her end is free to interact with Ku. We find that one Ku molecule dissociat es from I-site DNA with simple Langmuir (i.e., independent) kinetics. Howev er, two Ku molecules associate and dissociate from 2-site DNA with a time c ourse that cannot be described as a simple Langmuir interaction. On 3- and 4-site DNA, EMSA and SPR studies do not reveal any cooperativity, suggestin g that the middle Ku does not exhibit cooperative interaction with the two Ku molecules bound at the DNA ends. These results indicate that Ku molecule s can demonstrate cooperative interaction, and this is influenced by their positions along the DNA.