Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer.
We have previously identified a protein factor, PEBP2 (polyomavirus enhancer-binding protein), in the nuclear extract from mouse NIH 3T3 cells which binds to the sequence motif, PEA2, located within the polyomavirus enhancer A element. Upon cellular transformation with activated oncogene c-Ha-ras, this factor frequently undergoes drastic molecular modifications into an altered form having a considerably reduced molecular size. In this study, the altered form, PEBP3, was purified to near homogeneity. The purified PEBP3 comprised two sets of families of polypeptides, alpha-1 to alpha-4 and beta-1 to beta-2, which were 30 to 35 kilodaltons and 20 to 25 kilodaltons in size, respectively. Both kinds of polypeptides possessed DNA-binding activities with exactly the same sequence specificity. Individual alpha or beta polypeptides complexed with DNA showed faster gel mobilities than did PEBP3. However, the original gel retardation pattern was restored when alpha and beta polypeptides were mixed together in any arbitrary pair. These observation along with the results of UV- and chemical-cross-linking studies led us to conclude that PEBP3 is a heterodimer of alpha and beta subunits, potentially having a divalent DNA-binding activity. Furthermore, PEBP3 was found to bind a second, hitherto-unnoticed site of the polyomavirus enhancer that is located within the B element and coincides with the sequence previously known as the simian virus 40 enhancer core homology. From comparison of this and the original binding sites, the consensus sequence for PEBP3 was defined to be PuACCPuCA. These findings provided new insights into the biological significance of PEBP3 and PEBP2.
Kamachi
Y
Y
Department of Biochemsitry, Kyoto University, Japan.
Ogawa
E
E
Asano
M
M
Ishida
S
S
Murakami
Y
Y
Satake
M
M
Ito
Y
Y
Shigesada
K
K
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D050659:Core Binding Factor Alpha 1 Subunit; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D011905:Genes, ras; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D015345:Oligonucleotide Probes; D011120:Polyomavirus / Q000235:genetics*; D011485:Protein Binding; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*
D050659:Core Binding Factor Alpha 1 Subunit; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; C493716:Runx2 protein, mouse; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D003850:Deoxyribonuclease I