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Browsing by Author "Subramanian, Vidya, author"

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    Effect of histone H3 E73D mutation on in vitro chromatin silencing
    (Colorado State University. Libraries, 2007) Colbert, Karen, author; Subramanian, Vidya, author; Thompson, Jeffrey, author; Luger, Karolin, author
    Organization of DNA into chromatin requires the presence of small basic proteins called histones. Core histones are highly conserved across various species. In addition to their function as DNA packing material, histones play an important role in the regulation of transcription, replication, and gene silencing. In particular, histone-mediated silencing is achieved via histone modifications and through interactions with suppressor proteins. In the yeast Saccharomyces cerevisiae, a single amino acid substitution (E73D) found within the conserved region of histone H3, has been shown to de-repress silencing at the telomeres and mating type loci in yeast (Thompson et al., 2003). It has been proposed that the substitution may shorten the amino acid side chain length enough to disrupt a necessary interaction at the H3-H4 interface within the nucleosome. Alternatively, the E73D mutation may affect the binding affinity of Sir3 for histone H3. Sir3 and Sir4, chromatin-associated repressor proteins, are known to mediate telomeric and mating loci silencing in S. cerevisiae (Grunstein et al., 1997). In order to test the effect of the E73D mutation on nucleosome stability in vitro, the crystal structure of nucleosomes containing the H3 mutant will be determined to ascertain the charge interactions between the amino acid residues within the vicinity of the substituted residue. In addition, binding studies will be carried out to investigate Sir protein binding to nucleosomes containing H3 E73D histones. Finally, analytical ultracentrifugation experiments will provide quantitative data on the in vitro dynamics of nucleosomal arrays comprised of nucleosomes containing H3 E73D.
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    Role of polyglutamylation in nucleosome assembly protein 1 (NAP1) function
    (Colorado State University. Libraries, 2008) Subramanian, Vidya, author; Luger, Karolin, advisor
    The organization of DNA into chromatin requires the systematic deposition of the histones onto the DNA template. Chromatin function requires the dynamic exchange of the histone components during replication and transcription. Deposition and exchange is mediated in part by a family of proteins generally referred to as histone chaperones. It has been shown recently that recombinant yeast NAP1 (yNAP1) is capable of promoting ATP-independent histone exchange and nucleosome sliding in vitro, and this ability is specifically attributed to the highly acidic C terminal tail of the protein. Drosophila NAP1 (dNAP1) has a shorter acidic C terminus than yNAP1. Preliminary data in the lab suggests that recombinant wild-type dNAP1 is incapable of this nucleosome dissociation. Native dNAP1 purified from Drosophila embryos, on the other hand, is capable of nucleosome dissociation. In this study we reveal the presence of a unique post-translational modification, polyglutamylation in native dNAP1, which restores the nucleosome dissociation function to recombinant dNAP1. We have also been able to identify two target sequences, as well as the number of glutamyl units associated with these modifications using mass spectrometric analysis (MALDI & MS/MS). The modification at the CTAD (C-terminal acidic tail domain) could compensate for the lesser amount of acidic amino acid in dNAP1 and may account for the gain in nucleosome dissociation function. The second polyglutamylation site is located at the NLS (Nuclear Localization Sequence) (based on the conserved core domain of yNAP1 and dNAP1).