The aims of the proposed work are: to test in detail the validity and to assess the generality of our recently proposed 3-domain model for the high molecular weight subset (i.e., HMG-1 and its homologs) of the High Mobility Group nonhistone chromatin proteins; and to obtain quantitative estmatges of thermodynamic paratmers for the interactions of these proteins and their domains with DNA, histones, and chromatin. HMG-1 and HMG-E, from chicken erythrocytes, and HMG-T, from trout testis, will be purified by our previously described phosphocellulose chromatography method, supplemented with our recently developed chromatography on immobilized histone h5. When isolating the proteins we will intentionally avoid exposing them to overt denaturing conditions. In more commonly used procedures the proteins are exposed to very acidic conditions which denature them and from which they may not fully return to their native states. The proteins' domain structure will be studied by limited proteolysis, isolation of fragments (domains) released in protease treatments, and investigation of the physicochemical properties of the purified fragments. Interactions of HMG-1 and HMG-E and of their domains with DNA will be studied by fluorescence titration. Interactions of HMG-1 with histones will be studied by analytical ultracentrifugation, and interactions of HMG-1 with nucleosomal chromatin particles will be investigated with a gel permeation approach. The proteins under investigation are likely involved in transcription and/or DNA replication in nuclei of eukaryotic cells. Hence, our work should contribute to furthering our understanding of these important process, which are very incompletely understood in eukaryotes at the present time. In the long term, a full understanding of the dynamics and regulation of transcription and DNA replication in normal cells should lead to insights into aberrant regulation of those processes in disease states.
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| Abdul-Razzak, K K; Garg, L; Wen, L et al. (1987) Fetal and newborn calf thymus as a source of chromatin proteins: purification of HMG-1 and HMG-2. Prep Biochem 17:51-61 |
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