-
__ 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.
-
__ sometimes occur in DNA protein interactions & is more similar to RNA if it makes a double strand structure
- A-form
-
__ 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+).
-
__ has the largest diameter & __ has the smallest diameter.
- A DNA (A-form); Z DNA
-
__ has the largest distance per complete turn & rise per base pair and __ has the smallest distance per complete turn & rise per base pair.
- Z DNA; A DNA (A-form)
-
number of base pairs per complete turn: B DNA __, A DNA __, Z DNA __
10, 11, 12
-
In __ DNA, in purine & pyrimidine alternates, the __ is in anti & __ is in syn.
- Z; pyrimidine; purine
-
In __, DNA the position is always in anti conformation.
- B-DNA
-
syn conformation → __
left hand helix
-
anti-syn alternates → __
zigzag appearance
-
denaturation
- separating the 2 strands of DNA from each other
- can happen by breaking hydrogen bonds by high temperature or high pH
-
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.)
-
Absorption of UV light is by the __
bases
-
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)
- 40% less
-
Increasing temperature will cause a rapid __ in absorption.
- increase
-
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
-
What factors decrease the melting temp of DNA?
shorter DNA fragments = lower melting temp
-
Chromosomes of eukaryotes are __
long linear DNA molecules plus their associated proteins
-
Chromosomes of most of __ are circular.
- - prokaryotes, genetic materials of some viruses, DNA of mitochondria, and plasmids
-
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)
-
If the linking number (LK) = twist number (Tw) – Wr (writhe number), __
linking number is less than no writhe “relaxed.” Supercoil is negative.
-
If the linking number (LK) = twist number (Tw) + Wr (writhe number), __
linking number is greater than no writhe “relaxed.” Supercoil is positive
-
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°
-
DNA in __ can be unwind (separation of two strands) easier than __ DNA.
negative supercoil; relaxed
Organisms keep the DNA in negative
-
The thermophile bacteria living in hot springs have __.
positive supercoil
(preventing denaturation of DNA in the very high temperature of theenvironment )
-
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.
-
__ 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.
- Type I topoisomerases
-
__ 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
- Type II topoisomerases
-
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
-
2 circular DNA can __ with each other especially during the replication of circular DNA
- catenate
-
__ can decatenate (separate) 2 circular DNA
Topoisomerase II
- (If there is already a nick in 1 DNA, topoisomerase I also can decatenate them.)
-
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)
-
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
-
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
-
DNA topoisomers
circular DNA of the same size by different linking number
-
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
-
Negative supercoiled + EtBr →
more relaxed → slower movment
-
Less negative supercoiled + EtBr →
going beyond the relaxed to become positive supercoiled → fast movment
|
|
