Enhancer binding protein (EBP1) makes base and backbone contacts over one complete turn of the DNA double helix.
The simian virus 40 (SV40) enhancer consists of multiple DNA sequence motifs that represent the binding sites for a large number of trans-acting factors. We have purified one such factor, EBP1, which binds to a region encompassing the "core" of the SV40 enhancer, and appears to be involved in transcriptional activation. The interaction of EBP1 with its recognition site has been analysed by nuclease protection and by a variety of chemical probes. Enhancer sequences protected from cleavage with DNase I in the presence of EBP1 extend from position 232 to 250 on one strand and from 233 to 251 on the other strand. Methylation protection and alkylation interference studies have identified purine bases and backbone phosphate groups that participate in the formation of a specific EBP1-DNA complex. Within a ten base-pair region, every purine base interferes with binding when methylated and six phosphate groups on each strand interfere with binding when the attached oxygen groups are ethylated. "Footprinting" with hydroxyl radicals, generated by the 1,10-orthophenanthroline-copper ion, revealed sugar residues in the binding site that were protected from cleavage in the presence of EBP1. Computer graphics analyses of the contact point data indicate that EBP1 makes base and backbone contacts with the DNA over one complete turn of the DNA double helix, and suggest a model in which EBP1 makes sequence-specific contacts in the major groove, although binding may be influenced by interactions in the minor groove. Comparison of the EBP1 contact points with that of other known DNA-binding proteins indicates that EBP1 employs a unique mechanism to recognize a specific DNA sequence.
Clark
L
L
Department of Biochemistry and Microbiology, University of St. Andrews, Fife, Scotland.
Nicholson
J
J
Hay
R T
RT
D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D013539:Simian virus 40
D002352:Carrier Proteins; D010618:Phenanthrolines; D010710:Phosphates; D004247:DNA; D003850:Deoxyribonuclease I; C025205:1,10-phenanthroline