Topic 3 (DNA Structure and Topology) Flashcards
Where do purines attach to the ribose sugar?
C1, N9 of the base
Where do pyrimidines attach to the ribose sugar?
C1, N1 of the base
Which two interactions stabilize the structure of DNA?
Hydrogen bonds between the bases and pi-stacking of the bases
What effect does pi-stacking have that stabilizes the structure of DNA?
Hydrophobic effect and Van der Waals interactions
What is the handedness of a B-form DNA double helix?
Right-handed
What is the handedness of an A-form DNA double helix?
Right-handed
What is the handedness of a Z-form DNA double helix?
Left-handed
What form of the dNTPs is found in B-form DNA (anti or syn)?
Anti
What form of the dNTPs is found in Z-form DNA (anti or syn)?
Syn
Which direction does right-handed DNA spin?
Clockwise
What is the tautomer of an amide?
Imide
What is the tautomer of an enol?
Keto
What is tautomerization?
The migration of a hydrogen atom between areas on the same molecule
Define a tautomeric shift
The spontaneous rearrangement of nitrogenous bases that allow for hydrogen bonding of mismatch pairs
If a T tautomerized into its enol form, what would it pair with?
G
If a C tautomerized into its enol form, what would it pair with?
A
Base flipping allows for:
Homologous recombination and DNA repair
Explain the Mica experiment
A strand of DNA was attached to mica and exposed to DNase I. The enzyme would make a cut in the strand not attached to the mica every 10 nucleotides or so
What three strategies were used in the Mica experiment?
Affinity (attachment of DNA to mica), restriction digest, and gel electrophoresis
What conclusions could be made from the Mica experiment?
The DNA makes a 360 degree rotation every ~10.5 nucleotides, or every nucleotide is twisted 36 degrees from the previous one
What is the length of a B-DNA strand relative to the other forms?
Normal
What is the length of a A-DNA strand relative to the other forms?
Short
What is the length of a Z-DNA strand relative to the other forms?
Long
How many base pairs per helical turn for B-DNA?
10.5
How many base pairs per helical turn for A-DNA?
11
How many base pairs per helical turn for Z-DNA?
12
How many degrees per residue for B-DNA?
36
How many degrees per residue for A-DNA?
33
How many degrees per residue for Z-DNA?
30
True/False? Diffraction pattern lines are rotated 45 degrees to the actual lines formed by the DNA
False. They are perpendicular
What is pitch?
The length between rotations, 34 Angstroms
What is rise?
The distance between each nucleotide, 3.4 Angstroms
What is the radius of a DNA double helix?
10 Angstroms
When B-form DNA was used for x-ray diffraction, why did Franklin and Gosling conclude that DNA was a double helix?
Spot 4 was missing due to destructive interference because the phosphate backbones overlapped with each other
Why is the “X” formed in photograph 51 not formed by perpendicular lines?
Major and minor grooves
Define denaturation of DNA
The disruption of hydrogen bonds by heat, pH, or [salt]
Where can Tm (melting temperature) be found on a Temperature vs. Absorbance graph?
The midway point between where the absorbance changes from what you’d expect to see in double-stranded DNA to what you’d expect to see in single-stranded DNA (half of the DNA is denatured)
What does Tm represent?
The temperature at which the sample consists of 50% dsDNA and 50% ssDNA
Which factors affect Tm?
G:C content
Ionic strength of the solution
What effect does increasing G:C content have on TM? Why?
Increase; 3 hydrogen bonds require more energy than 2 (A:T), and G:C have lower entropy
What effect does increasing salt content have on TM? Why?
Increases in [salt] stabilize the phosphate backbone by decreasing the repulsive force between the two backbones, so dsDNA is more stable than usual
What absorbance does DNA absorb UV?
260nm
Describe the hyperchromic effect
ssDNA absorbs >40% of UV at 260nm than dsDNA due to dsDNA’s base stacking
What effect does base stacking have on UV absorbance?
Decreases absorbance
How does hybrid dsDNA occur?
When re-annealing, ssDNA can associate with another strand that has a similar sequence to form hybrid DNA
When 2 different but similar dsDNAs are denatured and re-annealed, what are the possible outcomes?
Could result in 2 hybrid strands or 2 normal strands
What experimental methods is DNA hybridization important for?
Southern blots, Northern blots, DNA and RNA microarrays, next-gen sequencing, identification of mutations
What is cccDNA?
Covalently closed, circular DNA
What is an example of cccDNA?
Bacterial plasmids
What can linear DNA do that circular DNA can’t?
Can freely rotate and unwind
During transcription, which end of the DNA is overwound? Why?
5’ end of the strand that is being transcribed. This is because as the DNA unwinds so RNA pol can transcribe, extra torsional stress is put on the end that it moves towards
During transcription, which end of the DNA is underwound? Why?
3’ end of the strand that is being transcribed. This is because as the DNA unwinds so RNA pol can transcribe, torsional stress is released from the end it moves away from
What is the formula for linking number (Lk)
Twist (Tw) + Writhe (Wr)
What does one twist and one writhe count as in terms of linking number?
Both -1
Define linking number
An integer representing the number of times it takes for a strand of DNA to pass through the other strand in order for the two strands to be entirely separated
True/False? Topoisomers differ in both linking number and base order
False. Only linking number
Using gel electrophoresis, how can we tell which band represents the relaxed coil or the supercoil? Why?
The smaller band is the supercoil as it is more compact and thus has less frictional resistance
List the order in which you would find a linear piece of DNA, supercoil with a Lk of -5, relaxed coil, and a supercoil with a Lk of -2 resulting from gel electrophoresis from largest to smallest
Relaxed, linear, supercoiled (-2), supercoiled (-5)
True/False? Supercoiling is determined by writhe, not twist
True
Which type of supercoil (positive or negative) is human DNA usually found in? Why?
Negative; It requires less energy to unwrap which is necessary for regular gene transcription
Which type of supercoil (positive or negative) is extremophile DNA usually found in? Why?
Positive; It requires more energy to unwrap (more stable), so DNA is less likely to be damaged or denatured by extreme environment
Define supercoiled DNA
DNA that twists upon itself because it is overwound or underwound (and thereby strained) relative to B-DNA
True/False? Topoisomerases can both introduce and relieve supercoils
True
Which direction must DNA be wound in to result in a negative supercoil?
Counterclockwise
Which direction must DNA be wound in to result in a positive supercoil?
Clockwise
A DNA strand has an Lk of -3 and is negatively coiled. What must be done to return the DNA to its relaxed form (Lk=0)?
A topoisomerase must break both strands of DNA, the DNA must then be rotated 360 degrees 3 times clockwise
A negatively supercoiled DNA (Lk=-3) must be unwound to its relaxed form (Lk=0). Explain the steps
Topoisomerase must break the DNA so one helix can rotate 3x360 degrees clockwise ***
How many times does a supercoiled DNA cross over itself if its Lk=-4? How many loops does it have?
Crosses over 4 times, has 5 loops
Which direction does a negative supercoil loop around a histone core?
Clockwise
Which direction does a positive supercoil loop around a histone core?
Counterclockwise
What are the functions of supercoiling?
To reduce the space and allow for DNA to be packaged into a small nucleus
To prevent or resolve DNA entanglement
Positive supercoiling protects DNA from thermal denaturation and regulates gene expression in extreme conditions
True/False? Negative supercoiling stores free energy required to facilitate strand compaction
False. Facilitates strand separation as negative coiling loosens up DNA winding
True/False? Topoisomerases do double-stranded breaks
False. Topoisomerase II does double, topoisomerase I does single stranded breaks
What is a type I topoisomerase?
It makes a ssDNA cut, does not require energy, and relaxes/introduces supercoils by 1 per digestion
Which amino acid residue on Topo I attacks the DNA?
Tyrosine (Tyr, Y)
Describe the steps performed by Topo I
DNA is nicked by attack of tyrosine
The other side of the cleaved DNA is held by the enzyme
The uncleaved strand passes through the opening
DNA ligation (no ATP required)
DNA is released
Why is energy not needed for Topo I to function?
Topo I is covalently linked to DNA after the nicking step. At the start of the reaction, one phosphate ester bond is broken and another one is formed while the strands rejoin, so the number of phosphate bonds remains the same
What is a type II topoisomerase?
Makes dsDNA cuts, requires energy, and reduces/introduces supercoils 2 per digestion
What is a common name for Topo II?
Gyrase
True/False? Gyrase can be found in both prokaryotes and eukaryotes
False. Only prokaryotes
What allows Topo I to do a catenation/decatenation?
ssDNA
What reactions is Topo II useful for?
Catenation/decatenation of dsDNA, and separating entangled DNA after replication
As the length of a reaction between topoisomerase and DNA goes on, what happens? What do we see in gel electrophoresis?
Lk decreases, “supercoiled” band moves closer and closer to the “relaxed” band
What is ethidium bromide (EtBr)?
A carcinogenic dye used to stain DNA which intercalates between stacked nucleotide bases, decreasing the angle per base from 36 degrees to 10 degrees
Why is EtBr carcinogenic?
Because it opens up the DNA, it is more susceptible to radiation/mutations
True/False? EtBr increases the length of DNA
True
