Nucleosome site exposure within oligonucleosomes

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DESCRIPTION (provided by applicant): This project will characterize the uncatalyzed site exposure and catalyzed nucleosome translocation within oligonucleosomes that is critical for understanding the unresolved question of how proteins access DNA within chromatin. First, the site exposure of DNA in a test nucleosome within an oligonucleosome will be measured by the restriction enzymes kinetics method. Second, the induced conformational changes of and binding rates to a FRET pair labeled test nucleosome within an oligonucleosome will be measured as the site-specific DNA binding protein, LexA, binds to the nucleosome. Steady state FRET signals will quantify conformational changes and FRET signals during stopped flow experiments will determine binding rates. Additional experiment will be done with H1, core histone mutants and changes in the oligonucleosome design to determine their affects on site exposure, protein binding rates and protein binding induced conformational changes. Finally, FRET measurements will determine if the chromatin remodeling protein, ISWl, increases the site exposure-opening rate and to learn what are the conformational changes induced during one ATP catalytic cycle. These results are critical for understanding how ISWl remodels chromatin.