Cleavage sites for the restriction enzymes are indicated by arrows. Shown are expected lengths of the single-stranded DNA fragments generated by cleavage with the restriction endonucleases AflIII, BstUI, and StyI. The positions of the nicks are indicated by asterisks. Ligation products included the fully ligated hairpin-ended DNA fragment ( hairpin) and partially ligated products containing a nick ( nicked hairpin). Two self-annealing oligonucleotides, separately purified on nondenaturing polyacrylamide gels, were ligated with T4-DNA ligase. A, schematic of the construction of the hairpin-ended DNA fragment f44-H. The data presented here support a model in which duplex DNA binds to the open channel, and a single-stranded DNA end is inserted into the enclosed cavity to activate the kinase.įigure 2 Construction of DNA fragment with hairpin ends. The structure of DNA-PK CS contains an open channel large enough for double-stranded DNA and an adjacent enclosed cavity with the dimensions of single-stranded DNA. Taken together, these data indicate that kinase activation involves a specific interaction with free single-stranded DNA ends. Significantly, short single-stranded oligonucleotides of 3–10 bases were capable of activating DNA-PK CS. Obstruction of DNA ends by streptavidin reduced both binding and activation of the kinase. When single-stranded loops were added to the DNA ends, binding was preserved, but kinase activation was severely reduced. The addition of unpaired single strands to blunt DNA ends increased binding and activation of the kinase. To define the DNA structure required for kinase activation, we synthesized a series of DNA molecules and tested their interactions with purified DNA-PK CS. The kinase is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PK CS. Glycobiology and Extracellular MatricesĭNA-dependent protein kinase (DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination.
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