Interactions of USF and Ku antigen with a human DNA region containing a replication origin.
By means of a combination of ion-exchange and sequence-specific affinity chromatography techniques, we have purified to homogeneity two protein complexes binding in a human DNA region (B48) previously recognized to contain a DNA replication origin. The DNA sequence used for the protein purification (B48 binding site) contains a binding site for basic-helix-loop-helix DNA binding proteins. The first complex is composed of two polypeptides of 42- and 44-kDa; its size, heat stability, and target DNA sequence suggest that it corresponds to transcription factor USF; furthermore, the 42-kDa polypeptide is recognized by antibodies raised against 43-kDa-USF. The second complex is represented by equimolar amounts of two proteins of 72 and 87 kDa; microsequencing of the two species indicated that they correspond to the human Ku antigen. In analogy with Ku, they produce a regular pattern of footprints without an apparent sequence-specificity, and their binding can be competed by unspecific DNA provided that it contains free ends. The potential role of B48 binding site and of these cognate proteins in origin activation is discussed.
Tóth
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EC
International Centre for Genetic Engineering and Biotechnology (ICGEB), AREA Science Park, Trieste, Italy.
Marusic
L
L
Ochem
A
A
Patthy
A
A
Pongor
S
S
Giacca
M
M
Falaschi
A
A
D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D051720:Upstream Stimulatory Factors
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen