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Protein and DNA interaction

Protein and DNA interaction. How to detect nucleosome position Xuefeng Zhu Department of Medical Biochemistry and Cell Biology 2009-11-17. Nucleosome and Chromatin. contents. What is the nucleosome? How the people discover the nucleosome? micrococcal nuclease cleavage

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Protein and DNA interaction

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  1. Protein and DNA interaction • How to detect nucleosome position Xuefeng Zhu • Department of Medical Biochemistry and Cell Biology • 2009-11-17

  2. Nucleosome and Chromatin

  3. contents • What is the nucleosome? • How the people discover the nucleosome? • micrococcal nuclease cleavage • Nucleosome and transcription • How to get nucleosome position?

  4. What is the nucleosome? DNA structure within the cell • DNA is associated with proteins in cells, both prokaryotes and eukaryotes, even viruses. DNA and its associated proteins is called a chromosome

  5. What is the nucleosome? Levels of chromatin structure

  6. What is the nucleosome? Nucleosome • Nucleosome is the basic block (lowest level) of chromatin organization. • Most of genomic DNA is confined in nucleosomes (one nucleosome per 146 base pairs on average). • Nucleosome core particle consist of histone octamer (two molecules each of H2A, H2B, H3 and H4 histone proteins) and super-coiled double-stranded DNA 125-166 base pairs long.

  7. What is the nucleosome?

  8. What is the nucleosome? In vitro reconsistution of nucleosome

  9. How the people discover the nucleosome? The nucleosome, was described by Roger Kornberg in 1974. Chromatin structure; oligomers of the histones. Kornberg RD, Thomas JO. Science. 1974 May 24;184(139):865-8. No abstract available. Chromatin structure: a repeating unit of histones and DNA. Kornberg RD. Science. 1974 May 24;184(139):868-71. No abstract available. 1975,Cambridge 2006,Standford

  10. How the people discover the nucleosome? Two types of experiments led to Kornberg's proposal of the nucleosome model. First, partial digestion of chromatin with micrococcal nuclease (an enzyme that degrades DNA) was found to yield DNA fragments approximately 200 base pairs long. In contrast, a similar digestion of naked DNA (not associated with proteins) yielded a continuous smear of randomly sized fragments. These results suggested that the binding of proteins to DNA in chromatin protects regions of the DNA from nuclease digestion, so that the enzyme can attack DNA only at sites separated by approximately 200 base pairs. Consistent with this notion, electron microscopy revealed that chromatin fibers have a beaded appearance, with the beads spaced at intervals of approximately 200 base pairs. Thus, both the nuclease digestion and the electron microscopic studies suggested that chromatin is composed of repeating 200-base-pair units, which were called nucleosomes.

  11. micrococcal nuclease cleavage Micrococcal nuclease Micrococcal nuclease is an endonuclease that cleaves DNA; in chromatin, DNA is cleaved preferentially between nucleosomes. Micrococcal nuclease Nucleosome

  12. micrococcal nuclease cleavage Microccocal nuclease initially cleaves between nucleosomes Microccocal nuclease initially cleaves between nucleosomes. Mononucleosomes typically have ~200 bp DNA. End-trimming reduces the length of DNA first to ~165 bp, and then generates core particles with 146 bp.

  13. micrococcal nuclease cleavage Micrococcal nuclease digestion pattern Micrococcal nuclease digests chromatin in nuclei into a multimeric series of DNA bands that can be separated by gel electrophoresis.

  14. Who decides nucleosome position? • Is nucleosome position random? • Does it depend on the DNA sequence? It majorly depends on DNA sequences, But not all.

  15. How does DNA sequence contribute to nucleosome positioning? • significant periodicities of dinucleotides and that DNA bending or flexibility helps in determining nucleosome position. AA/TT/TA Dinucleotides model Backbone inward Backbone out • ~60% of predicted high occupancy sites confirmed in vivo.

  16. Nucleosome and transcription What is function of nucleosome positioning? Nucleosome position depends on coding region DNA sequnces and couples to transcription

  17. Nucleosome and transcription Nucleosome positioning is couple to transcription activity. Coding region

  18. How to get nucleosome position? How to get nucleosome postion? Example: Yeast

  19. Experimental work How to get nucleosome position? Yeast culture Bioinformatic Analysis Raw data from tiling array Affymetrix tiling array software Normlization & calculation P Value & signal Export Visualize in Integrated Genome Browser

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