Mammalian β-Polymerase Promoter: Large-Scale Purification and Properties of ATF/CREB Palindrome Binding Protein from Bovine Testes

Steven Widen, Samuel H. Wilson

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

The mammalian DNA repair enzyme β-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 β-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 vs log KCl plots) and has a KA at 200 mM KCl of 5.8 × 1011 M-1. Kinetic studies with synthetic oligonucleotides as binding ligands indicate that the purified protein can bind tighter to or discriminate between the β-polymerase ATF/CREB element and similar elements derived from somatostatin and chorionic gonadotropin genes.

Original languageEnglish (US)
Pages (from-to)6296-6305
Number of pages10
JournalBiochemistry
Volume30
Issue number25
DOIs
StatePublished - Jun 1 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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