Chromatin associated proteins of Ustilago maydis
The eukaryotic genome is organized into chromatin through the specific association of histones, nonhistone chromosomal proteins, DNA topoisomerases and DNA. This nucleoprotein complex is as much a structural frame work for DNA metabolism as it is a regulator of its function. The role played by histones and nonhistone chromosomal proteins, from the lower eukaryote Ustilago maydis, in maintaining the structure and function of DNA is reported in this thesis. Fungal chromatin has been, thus far, inferred by the characterization of purified histones and chromatin associated proteins that participate in structural organization of the genome. To extend these observations, we developed a cell-free extract from the fungus Ustilago maydis in order to study the mechanism of chromatin assembly. The extract catalyzes DNA supercoiling of relaxed plasmid DNA template under physiological conditions, a result accepted as evidence of chromatin assembly. Biochemical and molecular experimentation confirmed that the supercoiling was due to the assembly of nucleosomes and also identified type I topoisomerase as the key enzyme involved in this process. A top1-strain, in which the type I topoisomerase gene of U. maydis is inactivated, was found to be deficient in promoting chromatin assembly in vitro, confirming the role of type I topoisomerase in this reaction. Exogenously added type I and II topoisomerase were able to regenerate the supercoiling reaction. The components of chromatin also include highly abundant nonhistone chromosomal proteins. These proteins have been shown to perform various functions during DNA metabolism. Nonhistone chromosomal proteins were initially identified as acid soluble, chromatin associated, High Mobility Group proteins (HMG). One such protein purified from U. maydis is High Mobility Protein 1 (Hmp1) which stimulates type I topoisomerase and binds DNA with secondary structure and mismatches. These activities were determined by in vitro assays and have suggested that Hmp1 may function in various DNA metabolic events. Transcription, repair, recombination and chromatin assembly are the most common metabolic activities of DNA and Hmp1 may be a general modulator of these events by facilitating the processing of DNA. A functional role for Hmp1 as a DNA 'chaperone' is proposed based on these interactions of the protein with DNA.
Dutta, Seema, "Chromatin associated proteins of Ustilago maydis" (1996). ETD Collection for Thomas Jefferson University. AAI9625287.