Sequence-specific binding of Ku autoantigen to single-stranded DNA.
Glucocorticoid-induced transcription of mouse mammary tumor virus is repressed by Ku antigen/DNA-dependent protein kinase (DNA-PK) through a DNA sequence element (NRE1) in the viral long terminal repeat. Nuclear factors binding to the separated single strands of NRE1 have been identified that may also be important for transcriptional regulation through this element. We report the separation of the upper-stranded NRE1 binding activity in Jurkat T cell nuclear extracts into two components. One component was identified as Ku antigen. The DNA sequence preference for Ku binding to single-stranded DNA closely paralleled the sequence requirements of Ku for double-stranded DNA. Recombinant Ku bound the single, upper strand of NRE1 with an affinity that was 3-4-fold lower than its affinity for double-stranded NRE1. Sequence-specific single-stranded Ku binding occurred rapidly (t1/2 on = 2.0 min) and was exceptionally stable, with an off rate of t1/2= 68 min. While Ku70 cross-linked to the upper strand of NRE1 when Ku was bound to double-stranded and single-stranded DNAs, the Ku80 subunit only cross-linked to single-stranded NRE1. Intriguingly, addition of Mg2+ and ATP, the cofactors required for Ku helicase activity, induced the cross-linking of Ku80 to a double-stranded NRE1-containing oligonucleotide, without completely unwinding the two strands.
Graduate Program in Biochemistry, University of Ottawa, Loeb Institute for Medical Research, Ottawa Civic Hospital, Ottawa, Ontario K1Y 4E9, Canada.
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Torrance
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Giffin
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Rodda
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Pope
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Haché
D034961:Antigens, Nuclear; D001324:Autoantigens / Q000378:metabolism*; D001483:Base Sequence; D018983:DNA Footprinting; D004265:DNA Helicases; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D000072200:Ku Autoantigen; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic
D034961:Antigens, Nuclear; D001324:Autoantigens; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen