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__ is the average conformation (of a double helix) seen in physiological conditions
B-form
- Depending on the sequence of DNA, the B-form may change to some extent affecting the major and minor grooves.
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__ sometimes occur in DNA protein interactions & is more similar to RNA if it makes a double strand structure
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__ is a left handed helix with a zigzag structure
- Z DNA
- This happens if the sequence is made of purine and pyrimidine alternates in a solution with high concentration of charged ions (Na+).
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__ has the largest diameter & __ has the smallest diameter.
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__ has the largest distance per complete turn & rise per base pair and __ has the smallest distance per complete turn & rise per base pair.
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number of base pairs per complete turn: B DNA __, A DNA __, Z DNA __
10, 11, 12
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In __ DNA, in purine & pyrimidine alternates, the __ is in anti & __ is in syn.
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In __, DNA the position is always in anti conformation.
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syn conformation → __
left hand helix
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anti-syn alternates → __
zigzag appearance
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denaturation
- separating the 2 strands of DNA from each other
- can happen by breaking hydrogen bonds by high temperature or high pH
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The two strands can be attached with each other again (renaturation) if __. This reattachment is by restoration of __ and is known as __
- The temperature or pH are brought down;
- hydrogen bonds;
- hybridization or annealing
(For this hybridization the sequences of both strands should be complementary.)
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Absorption of UV light is by the __
bases
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In the double strand DNA, due to the stack of bases that makes them unavailable to UV, the UV absorption is __ compared to 2 separate single strands of DNA (denatured DNA)
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Increasing temperature will cause a rapid __ in absorption.
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What factors increase the melting temp of DNA?
- higher G:C = more hydrogen bonds = higher melting pointAlso in base pair stacks, G:C has more interaction with adjacent base pairs.
- ionic strengths: higher salt concentration = higher melting temp. High levels of positively charged ions surround the negatively charged phosphates in DNA backbone, therefore its is easier for both strands to not repel each other
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What factors decrease the melting temp of DNA?
shorter DNA fragments = lower melting temp
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Chromosomes of eukaryotes are __
long linear DNA molecules plus their associated proteins
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Chromosomes of most of __ are circular.
- - prokaryotes, genetic materials of some viruses, DNA of mitochondria, and plasmids
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A circular DNA can be generated by __
- covalently joining both ends of a DNA molecule (covalently closed circular DNA (cccDNA)
- Circular DNA is in topological constrict (cannot very freely rotate)
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If the linking number (LK) = twist number (Tw) – Wr (writhe number), __
linking number is less than no writhe “relaxed.” Supercoil is negative.
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If the linking number (LK) = twist number (Tw) + Wr (writhe number), __
linking number is greater than no writhe “relaxed.” Supercoil is positive
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DNA can be long or short which changes the LK value, thus to normalize the LK, the __ is calculated.
- super helical density (σ)
- σ = ΔLK / LK°
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DNA in __ can be unwind (separation of two strands) easier than __ DNA.
negative supercoil; relaxed
Organisms keep the DNA in negative
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The thermophile bacteria living in hot springs have __.
positive supercoil
(preventing denaturation of DNA in the very high temperature of theenvironment )
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The supercoil can be resolved by making a temporary break in__ bond(s) of one or both DNA strand(s). This is done usually by __ enzymes.
- phosphodiester;
- topoisomerase
- After the release of the supercoil (decreasing LK) the phosphodiester bond is restored.
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__ introduce a break in one strand’s phosphodiester bond (a nick) and pass the second strand through the generated gap. The two ends of thebroken strand are then re-aligned.
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__ break both strands of the double helix, creating a “gate” through which a second segment of the double helix is passed. Requires the use of ATP
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Bacteria also have the enzyme __ which is a type of topoisomerase. Instead of relaxing the DNA, it induces negative supercoil which is favorable for DNA replication and transcription.
gyrase
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2 circular DNA can __ with each other especially during the replication of circular DNA
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__ can decatenate (separate) 2 circular DNA
Topoisomerase II
- (If there is already a nick in 1 DNA, topoisomerase I also can decatenate them.)
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Linear chromosomes of eukaryotes can get entangled especially during DNA replication. __ untangles 2 newly made DNA from each other.
Topoisomerase II
- (can also remove knots. If DNA has a nick, then topo I can also undo the knot)
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During the activity of topoisomerases, one end of each cut polynucleotide becomes covalently attached to a __ at the active site of the enzyme (held tightly in place) while the __ is being manipulated. This assures __. Then open __ on the other side of the cut attacks the __ restoring the bond and realigning the 2 pieces of DNA together
- tyrosine amino acid;
- free end;
- to rejoin the strand and prevents a permanent cut in the DNA;
- hydroxyl;
- phosphotyrosine
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steps of topo enzyme activity
- 1. cleavage of DNA
- 2. strand passage
- 3. DNA rejoining
- Topo binds to the melted DNA cleavage by topo and phosphotyrosine formation (covalent bond of enzyme & DNA) and holding the other side of broken DNA strand. Change of enzyme conformation and moving the intact strand through the broken one.
- Change of conformation again and rejoining the 2 sides of broken DNA (no change in DNA sequence but change in supercoiling). The same mechanism for topo II except the enzyme is a dimer or tetramer and makes cuts on both strands
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DNA topoisomers
circular DNA of the same size by different linking number
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Relaxed circular DNA moves __ in gel electrophoresis than linear DNA. Supercoiled DNA moves __.
- slower;
- faster (as it is more compact)
- The more supercoiled, the faster it moves
-
EtBr
- intercalates between bases and unwinds them
- decreases the twist of DNA (from 36 degrees to 10 degrees per base pair)
- decreases the negative supercoil (Wr is increased), makes it close to 0 & if more EtBr is added, the DNA becomes positively supercoiled.
- affects movement of cccDNA in the gel
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Negative supercoiled + EtBr →
more relaxed → slower movment
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Less negative supercoiled + EtBr →
going beyond the relaxed to become positive supercoiled → fast movment
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