Activation of DNA-dependent protein kinase by single-stranded DNA ends.
DNA-dependent protein kinase (DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination. The kinase is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PK(CS). 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 addition of unpaired single strands to blunt DNA ends increased 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. Obstruction of DNA ends by streptavidin reduced both binding and activation of the kinase. Significantly, short single-stranded oligonucleotides of 3-10 bases were capable of activating DNA-PK(CS). Taken together, these data indicate that kinase activation involves a specific interaction with free single-stranded DNA 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. 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.
Hammarsten
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Department of Medicine, Stanford University School of Medicine, Stanford, California 94305-5115, USA.
DeFazio
L G
LG
Chu
G
G
D034961:Antigens, Nuclear; D001667:Binding, Competitive / Q000187:drug effects; D003461:Crystallography; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D004277:DNA, Single-Stranded / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D007202:Indicators and Reagents / Q000494:pharmacology; D007700:Kinetics; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000378:metabolism; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019809:Streptavidin / Q000494:pharmacology
D034961:Antigens, Nuclear; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D009687:Nuclear Proteins; D004247:DNA; D019809:Streptavidin; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen