Mammalian beta-polymerase promoter: large-scale purification and properties of ATF/CREB palindrome binding protein from bovine testes.
The mammalian DNA repair enzyme beta-polymerase is encoded by a single-copy gene that is expressed in all tissues and cell lines studied to date. A protein fraction with high binding affinity for an ATF/CREB-like binding element, GTGACGTCAC, at -49 to -40 in the core beta-polymerase promoter has been purified to near-homogeneity from a nuclear extract of bovine testes. The major binding activity, as monitored by gel mobility shift assay, is recovered in 20% yield by a procedure involving oligonucleotide affinity chromatography. The purified protein yields DNase I footprinting and gel shift binding patterns indistinguishable from the activity in crude extracts. The final fraction activates transcription in an in vitro transcription reaction. The native molecular weight of the purified binding activity is about 100-120K as measured by gel filtration. SDS-PAGE of the purified fraction revealed that it contains several polypeptides in the molecular weight range of 30-52K, yet two of these peptides (Mr 49K and 52K) are predominant. Specific binding to the palindrome is salt-sensitive and is consistent with the formation of nine ion pairs (from log KA vs log KCl plots) and has a KA at 200 mM KCl of 5.8 X 10(11) M-1. Kinetic studies with synthetic oligonucleotides as binding ligands indicate that the purified protein can bind tighter to or discriminate between the beta-polymerase ATF/CREB element and similar elements derived from somatostatin and chorionic gonadotropin genes.
Widen
S G
SG
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Wilson
S H
SH
D051696:Activating Transcription Factors; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001798:Blood Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D017362:Cyclic AMP Response Element-Binding Protein; D004256:DNA Polymerase I / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013737:Testis / Q000737:chemistry / Q000201:enzymology*; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D051696:Activating Transcription Factors; D001798:Blood Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004256:DNA Polymerase I