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What is topology?
Spatial properties that are preserved under continuous deformations of objects
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What is supercoiling
- Coiling of a coil, is only applied if both strands remain covalently intact and tension is stored. Closed-circular DNA is a good example. If a break occurs in any strand, torsional strains and supercoils are removed.
- If DNA is underwound, a negative supercoil will form. If DNA is overwound, a positive supercoil will form.
- Cellular DNA is underwound (5-7%) and negatively supercoiled.
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Explain linking number
- Linking number is a useful way to describe the topological properties of DNA.
- It is the number of times one strand of DNA winds around the other.
- Only applied if both strands are covalently intact.
- LK0 = relaxed state in B form DNA = number of bp divided by 10.5 bp.
- Linking number is equal to twist + writhe
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Explain twist
Number of helical turns
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Explain writhe
Number of large distortions of the long axis of the helix
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What is the linking number of the 5250-bp plasmid isolated from E.coli
- LK0 is 5250/10.5 = 500. In a relaxed state, the plasmid has 500 helical twists.
- The image of the plasmid shows 4 supercoils (kesisim), because the plasmid came from a bacterial cell, these must be negative supercoils (as in all cells)
- So the LK is the LK0 - 4 = 500 - 4 = 496
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Why is DNA in cells supercoiled and underwound?
For compaction and strand separation.
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What are topoisomerases?
- This is how DNA becomes underwound in cells.
- Enzymes that regulate the topological state by changing the linking number. Topoisomerases can relax supercoiled DNA.
- Topoisomerases catalyze three steps:
- Cleavage of DNA strand
- Passing the intact strand through the break
- Resealing the strand
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Explain the different types of topoisomerases
- Topoisomerase 1 can change the linking number by breaking a single strand. Can change LK 1 at a time. Uses no ATP, instead relying on the internal tension of the DNA.
- Topoisomerase 2: can change LK in steps of 2. Breaks both DNA strands at the same time. Requires ATP to work.
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What is chromatin?
- DNA + tightly bound proteins
- There for organization and regulation
- Proteins called histones represent the major class of proteins in chromatin
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Explain the mechanism of action of bacterial type 1 topoisomerase
- The enzyme itself converts between the closed and open conformation
- Step 1 is cleaving: the tyrosine residue on the enzyme attacks the DNA backbone, and the enzyme goes from closed to open.
- Step 2 is strand passing: The actual step where LK changes.
- Step 3 is closing: the topoisomerase allows the DNA to religate and releases it.
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Explain why DNA replication generates a major topological problem
- When DNA is replicated, the progression of the replication fork creates overwinding of the DNA downstream. If not addressed, the energy required to continue opening the DNA would be too high to progress.
- Cells use topoisomerases to undo the positive supercoils that pop up downstream of the replication fork.
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Explain DNA gyrase
- Prokaryotic type 2 topoisomerase that specifically adds negative supercoils to DNA or relaxes positive supercoils.
- In other words, it always decreases LK, regardless of whether DNA is positively supercoiled, negative or neutral.
- Uses ATP
- Not in eukaryotes
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What is the nucleosome length?
200 bp + histone core on a 20 nm fiber.
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Explain histone core
- Histone core is an octomer containing 2 molecules of each of 4 core histones (H2A, H2B, H3, H4)
- Histones are small, positively charged proteins with high affinity to DNA
- Histones are highly conserved.
- DNA forms about 14 hydrogen bonds with the histone core particle, with all the interactions occurring in a sequence specific way in the minor groove between the oxygen atoms in the phosphodiester backbone and the protein.
- DNA bound to nucleosomes is compacted about 7 fold.
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How do eukaryotes (without gyrase) maintain their genomes in an underwound state?
- Eukaryotic topoisomerases cannot underwind DNA.
- Histone wrapping generates one negative supercoil.
- Because no nicking has occurred, a positive supercoil forms to compensate for this negative supercoil.
- The positive supercoil can be relieved by a topoisomerase but the negative supercoil cannot be relieved, leaving a net -1 supercoil.
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Explain regulation of chromatin structure
- The interaction of DNA and the histone octamer must be dynamic.
- Cellular processes must regulate conversion between the 30 nm and the 10 nm fiber and the exposure of DNA.
- This is done through histone modifications, which are covalent modifications that affect the net charge, shape and other properties of histones.
- These modifications produce a histone code.
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Give examples of histone modifications
- Phosphorylation, methylation, acetylation
- Lysine acetylation increases accessibility by removing positive charges on histone tails and affecting the ability to form the 30 nm fiber.
- Histone acetyl transferases interact with promoters to acetylate lysines, and then the transcriptional machinery binds promoters.
- Acetylation also helps recruit nucleosome remodeling complexes.
- Histone modifying and chromatin remodeling complexes work together to alter chromatin.
- Deacetylase helps to close the chromatin.
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