Increased DNA-bending Activity and Higher Affinity DNA Binding of High Mobility Group Protein HMG-1 Prepared without Acids (∗)

Recently, DNA ring closure assays showed that high mobility group protein HMG-1 and its close homolog HMG-2 mediate sequence-independent DNA flexion. This DNA-bending activity appears to be central to at least some of the recently elucidated functions of HMG-1/2, such as the enhancement of progesterone receptor DNA binding. Here we show that standard purification procedures utilizing perchloric and trichloroacetic acid can produce HMG-1 significantly deficient in its abilities to bind and bend double-stranded DNA, while acid-independent methods purify HMG-1 that is superior in these respects. Significant losses of DNA ring closure activity were seen upon limited 2-5-h exposures of non-acid-purified HMG-1/2 to perchloric acid and/or trichloroacetic acid. Measurements of the apparent DNA dissociation binding constant (Kd(app)) of acid-extracted preparations of HMG-1 gave a wide range of values, and only those preparations demonstrating little DNA ring closure activity had Kd values near the previously published value (∼10−6M). The highest ring closure activities and lowest Kd(app) (