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 nonacid-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 (approximately 10(-6) M). The highest ring closure activities and lowest Kd(app) (< 3 x 10(-9) M) were obtained for HMG-1 purified without acids. These combined results support the use of alternative, non-acid purification procedures for preserving the DNA-bending activity of HMG-1/2 and suggest that past procedures utilizing acids have led to an underestimation of the affinity of HMG-1 for DNA.
Wagner
J P
JP
Department of Biochemistry, University of Colorado Health Sciences Center, Denver 80262, USA.
Quill
D M
DM
Pettijohn
D E
DE
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D051381:Rats; D011980:Receptors, Progesterone / Q000378:metabolism; D013950:Thymus Gland / Q000378:metabolism
D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D011980:Receptors, Progesterone; D004247:DNA