The human Ku heterodimer has been shown to
act as the regulatory subunit of DNA-dependent protein kinase holoenzyme
(DNA-PK). Different cell lines have been identified as defective in one
of the DNA-PK components. Theses cells are highly sensitive to X-ray
irradiation and also severely deficient in the ligation of reaction
intermediates generated during V(D)J recombination. The yKu heterodimer
binds specifically to ends of dsDNA and to DNA discontinuities like
nicks and gaps in the DNA double helix. Recently, we have shown that yKu
is involved in DNA repair and recombination. Using an in vivo
plasmid rejoining assay, Boulton and Jackson could demonstrate that yKu
is essential in the repair of DNA double strand breaks bearing cohesive
ends. In wild type strains, this damage is repaired accurately. In yku
deficient strains, deletions of up to several hundred bp occur before
DNA ends can be ligated. Now, we can show that yKu acts as a DNA
helicase in vitro . YKu unwinds ds DNA with overhanging ends, but
cannot unwind blunt end DNA. YKu helicase proceeds in 5' - 3' direction
on the bound strand. Dephosphorylation of yKu using BAP results in a
dramatically decrease of helicase activity. To characterize the in
vivo function of yKu helicase activity we are generating mutants of
the ATP-binding motif of the p70 subunit. (Supported by the Deutsche
Forschungsgemeinschaft, Wi 319/11-3 project 7)
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