DNA binding specificities of YPF1, a Drosophila homolog to the DNA binding subunit of human DNA-dependent protein kinase, Ku.
YPF1, a heterodimeric protein from Drosophila melanogaster, is a homolog to Ku, the DNA binding subunit of human DNA-dependent protein kinase. This kinase is crucial in transcriptional activation, V(D)J recombination, double-strand break repair, and both topoisomerase and helicase activities. To investigate functional homology between YPF1 and Ku, we examined DNA binding properties of YPF1. Like Ku, at 100 mM KCl, YPF1 binding has no detectable DNA sequence specificity, requires a DNA terminus, and has a concentration-dependent stoichiometry consistent with subsequent translocation along DNA. YPF1 differs from Ku by having a 10(5)-fold higher affinity. At 400 mM KCl, YPF1 still prefers DNA termini but shows binding specificities not observed previously with Ku. In descending order of affinity, YPF1 binds to: specific DNA sequences with a specific polarity and spacing relative to DNA termini; nonspecific linear DNA; and circular DNA. At this higher ionic strength, binding stoichiometry is concentration independent, indicating that YPF1 remains bound to ends. These results demonstrate a strong functional homology between YPF1 and Ku at physiological ionic strength. The strong binding of YPF1 has also allowed us to detect underlying binding specificities that may be specific to YPF1 and its function.
Jacoby
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DB
Rosenstiel Center, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Wensink
P C
PC
D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000378:metabolism*; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009994:Osmolar Concentration; D011189:Potassium Chloride; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C086846:Irbp protein, Drosophila; D009687:Nuclear Proteins; D011189:Potassium Chloride; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen