Purification and characterization of a DNA-binding heterodimer of 52 and 100 kDa from HeLa cells.
In studies of protein binding to the upstream region of the human proliferation-associated antigen p120 gene, a heterodimer of 52 and 100 kDa proteins was purified from HeLa cells. A 1:1 ratio of p52 and p100 was constant throughout the purification. The heterodimer was localized to cell nuclei, as shown by immunofluorescence. The pI values of the p52 and p100 were 7.8 and 8.6 respectively. The peptide sequences obtained for p52 (QSNKTFNLEKQNHTPRKKHQ and PLRGKQLRVRFAAHSASLTVR) and for p100 (PGGPKPGGGPGLSTPGGHPKPPHRGGGEPPRGRQ and GPGPGQSGPKPPIPPPPPHQQ) were not found in the computer databanks. One p52 peptide sequence, PLRGKQLRVRFA, shows considerable sequence similarity to a conserved motif in topoisomerase II of multiple species. The p52/100 heterodimer bound to different DNA probes. The binding was competed by poly(dI-dC), sonicated salmon sperm DNA, and circular or linearized plasmid DNA. The optimal DNA binding for the heterodimer was at pH 7-9 with low salt. The DNA-binding subunit of the heterodimer was the p100 polypeptide, as shown by u.v.-cross-linking assays and Southwestern blots.
Zhang
W W
WW
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030.
Zhang
L X
LX
Busch
R K
RK
Farrés
J
J
Busch
H
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000235:genetics*; D009994:Osmolar Concentration; D012359:tRNA Methyltransferases
D015342:DNA Probes; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D004247:DNA; C065028:NOP2 protein, human; D012359:tRNA Methyltransferases