EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA.
We have previously reported the purification and characterization of the transcription factor EBP-80 (Falzon, M., and Kuff, E. L. (1989) J. Biol. Chem. 264, 21915-21922). EBP-80 mediates the DNA methylation effect on transcription from an endogenous proviral long terminal repeat. Here we show that EBP-80 is very similar if not identical to the Ku autoantigen, a heterodimeric nuclear protein first detected by antibodies from autoimmune patients (Mimori, T., Akizuki, M., Yamagata, H., Inada, S., Yoshida, S., and Homma, M. (1981) J. Clin. Invest. 68, 611-620). A number of laboratories have shown that the Ku protein complex binds to free double-stranded DNA ends. In this study, we have examined the binding properties of EBP-80. EBP-80 binds single-stranded DNA with low affinity. Binding to random sequence double-stranded DNA depends on the length of the duplex and is optimal with oligomers of 30 and 32 base pairs; the protein complexes formed with these oligomers have Kd values of 15-20 pM. It binds with comparable high affinities to blunt-ended duplex DNA, to duplex DNA ending in hairpin loops, and to constructs in which an internal segment of duplex DNA is flanked by single-strand extensions. EBP-80 also interacts effectively with circular duplex molecules containing a 30-nucleotide single-stranded region (gap) or a double-stranded segment of nonhomology (bubble), but only weakly with the corresponding closed circular construct made up entirely of duplex DNA. EBP-80 prefers A/T to G/C ends. The binding properties of EBP-80 are consistent with the hypothesis that is recognizes single- to double-strand transitions in DNA. A model is presented for the interaction of EBP-80 with its target sequence in the proviral long terminal repeat.
Falzon
M
M
Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892.
Fewell
J W
JW
Kuff
E L
EL
D000260:Adenoviruses, Human / Q000235:genetics; D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D004265:DNA Helicases; D004270:DNA, Circular / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000494:pharmacology; D012091:Repetitive Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D034961:Antigens, Nuclear; D001324:Autoantigens; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D014157:Transcription Factors; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen