Abstract
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.
Highlights
DNA ring closure assays showed that high mobility group protein HMG-l and its close homolog
The results of the present report are of significance in that they demonstrated that the most common purification protocols utilizing perchloric and trichloroacetic acid extraction can lead to inactivation of HMG-1 as measured by two important parameters, its ability to bind DNA with high affinity and its ability to flex double stranded DNA as measured by DNA ring closure assays
Short exposures to both perchloric acid (PCA) and trichloroacetic acid were shown to have significant detrimental effects on the ring closure activity of different non-acid-extracted preparations of HMG-1/2 (Fig. 1). These results are in agreement with several previous studies, undertaken before the DNA-bending activity of HMG-1/2 was known, which showed that these acids could affect other properties of HMG-1I2
Summary
DNA ring closure assays showed that high mobility group protein HMG-l and its close homolog. This DNA-bending activity appears to be central to at least some of the recently elucidated functions of HMG-. We show that standard purification procedures utilizing perchloric and trichloroacetic acid can produce HMG-l significantly deficient in its abilities to bind and bend double-stranded DNA, while acidindependent methods purify HMG-l that is superior in these respects. Values near The highest the previously pubring closure activities and lowest Kdlapp) «3 X 10-9 M) were obtained for HMG·l purified without acids These combined results support the use of alternative, non-acid purification procedures for preserving the DNA·bending activity of HMG-1I2 and suggest that past procedures utilizing acids have led to an underestimation of the affinity of HMG·l for DNA. There is evidence that this ability to alter DNA structure by bending may be important to at least some of the cellular functions of the protein
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
