Challenge: How could you tell if the topoisomerase is making positive or negative supercoils?
- Use 2D gel electrophoresis(different tool)
- EtBr
- Use the topoisomerase that can only relax negative supercoil
The type I?
This week’s learning objectives:
- Explain how the binding of proteins to DNA can affect DNA topology.
- Describe the structure of nucleosomes and give examples of how they are packaged into higher order chromatin structures.
- Analyze experiments that determine nucleosome positioning and organization in the cell.
Higher Order Chromatin Structure Involves Multiple Levels of Compaction
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Topoisomerase would like to access the supercoiled DNA to make them relaxed.
Why is genomic DNA negatively supercoiled?
- helps to package DNA
- makes DNA easier to unwind
Ways to induce negative supercoiling in vivo
Bacteria –
DNA gyrase
Eukaryotes –
nucleosomes + topoisomerases
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Design an Experiment to show that nucleosome assembly causes negative supercoiling
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- Incubate DNA with nucleosomes
- Add purified topoisomerases
- Topoisomerases will preferentially remove positive plectonemic supercoils
- Add SDS to inactivate the topoisomerases and remove the nucleosomes
- The negative supercoils will remain
But we do not know the supercoil is negative or postive just according to this experiment.
Nucleosomes are ~146 bp of DNA + 8 histone subunits
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Protein composition of a typical nucleosome
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Histone proteins are highlt basic.(positively charged)
Poll Everwhere
In SDS-PAGE, histones migrate more slowly than they should based on their molecular weight.
What could explain this?
Histone Binding with DNA
Histone proteins have non-specific interactions with oxygen atoms in the DNA backbone and with bases in the minor groove
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DNA sequence can affect nucleosome binding
The A-T base pair preference facilitates minor groove compression and assists with DNA bending
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Nucleosome assembly requires histone chaperones
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Histone tails extrude far from the nucleosome
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Histone tails can be modified in several ways
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Uniquitin is a small polypeptide
- Ubiquitin can be linked to itself, creating di-ubiquitin, tri-ubiquitin, and poly-ubiquitin chains
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Enzymes catalyze histone modifications
Acetyltransferase, deacetylase, methyltransferase
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Other Histone variants are found in specialized regions of the genome
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Determining if nuleosomes are present:micrococcal nuclease (MNase) digestion
MNase cuts preferentially between nucleosomes in the linker DNA
If you use a low concentration
of MNase, then cutting will occur randomly along the length of the genomic DNA fragments
(much like a DNase footprinting assay)
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Poll Everywhere
Here is a figure showing the results of MNase digestion of nucleosomal DNA
in the absence or presence of histone H1.
What do these results suggest that histone H1 does to chromatin?
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