Semi-Synthesis to Examine the Role of Phosphorylation of Histone H3 at T118
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Publisher:The Ohio State University
Series/Report no.:The Ohio State University. Department of Biochemistry Honors Theses; 2009
The basic unit of chromatin is the nucleosome; 147 bp of DNA wrapped around an octamer core of histone proteins. Post-translational modifications of these histone proteins have the potential to dramatically alter chromatin structure and dynamics, which play an important role in vital processes such as DNA repair and transcription. H3-T118 is located at the dyad region of the nucleosome at the histone-DNA interface, and directly interacts with DNA. Mutation of this residue shows weakened DNA contacts or lethality, and phosphorylation has been detected by mass spectrometry. We believe that phosphorylation of H3-T118 is likely to significantly weaken histone-DNA interactions. In order to examine this, we prepared semi-synthetic H3-pT118 using expressed protein ligation to join a recombinant H3 thioester with a modified synthetic peptide at a native cysteine residue. We refolded H3-pT118 with histone proteins H2A, H2B, and H4 into the histone octamer and reconstituted into nucleosomes using DNA of varying lengths, each containing a derivative of the 601 nucleosome positioning sequence. Native gel analysis shows the presence of altered structures, in addition to the expected mononucleosomes. Exonuclease III nucleosome positioning analysis shows the mononucleosomes are depositioned from the normal central position. The altered structure appears to be consistent with two octamers and two segments of DNA by sucrose gradient centrifugation, electrophoretic mobility, gel analysis, and atomic force microscopy. These observations suggest a unique role for the phosphorylation of H3-T118 in the regulation of chromatin structure.
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