A mammalian high mobility group protein recognizes any stretch of six A·T base pairs in duplex DNA

α-Protein is a high mobility group protein originally purified from African green monkey cells based on its affinity for the 172-base-pair repeat of monkey α-satellite DNA. We have used DNase I footprinting to identify 50 α-protein binding sites on simian virus 40 DNA and thereby to determine the DNA binding specificity of this mammalian nuclear protein. α-Protein binds with approximately equal affinity to any run of six or more A·T base pairs in duplex DNA, to many, if not all, runs of five A·T base pairs, and to a small number of other sequences within otherwise (A+T)-rich regions. Unlike well characterized sequence-specific DNA binding proteins such as bacterial repressors, α-protein makes extensive contacts within the minor groove of B-DNA. These and related findings indicate that, rather than binding to a few specific DNA sequences, α-protein recognizes a configuration of the minor groove characteristic of short runs of A·T base pairs. We discuss possible functions of α-protein and the similarities in DNA recognition by α-protein and the antibiotic netropsin.