Identification by Exonuclease Footprinting of a Distal Promoter-Binding Protein from HeLa Cell Extracts

Abstract

Incubation of a HeLa cell fraction termed P1000 with genomic DNA fragments containing portions of the 5′-flanking region of the ovalbumin gene, followed by digestion with exonucleases, shows that this cell fraction contains a protein (or proteins) that binds to the distal promoter of the ovalbumin gene. The protein protects both strands and spans a minimum of 22 bp on the noncoding strand from nucleotides −68 to −90. The 5′ border of the protected region on the coding strand is located at nucleotide −81. A similar site on the chick β-globin gene is also protected by HeLa cell fraction P1000. The exonuclease footprinting methods used to identify the distal promoter-binding protein are more sensitive than the DNasel footprinting method, and can be used to identify sequence-specific binding proteins in a mixture of DNA binding proteins.