Topic 3 Flashcards
GC is more stable than AT. True or false: this is predominantly due to the extra hydrogen bond in the GC base pair
False. This is a misconception.
- GC actually has stronger π stacking compared to AT which is the main contributor to GC’s stability
Most DNA is (right-handed/left-handed)
Right-handed
What does it mean for a DNA helix to be “right-handed”?
DNA turns in the direction of your right hand fingers curling inward (with your thumb pointed up)
- If you follow the helix as it twists away from you, the spiral moves in the clockwise direction.
Right-handed DNA results when the ribose and base are in (anti/syn) configuration
Anti (base is not oriented directly above the ribose)
Left-handed DNA results when the ribose and base are in (anti/syn) configuration
Syn (base is oriented directly above the ribose)
Tautomers/tautomerization
Isomeric molecules that can be interconverted by tautomerization, a chemical reaction that often results in the migration of a hydrogen atom
Amides tautomerize to…
Imides (imidic acid)
- Double bond between carbon and nitrogen, with alcohol group branching off the carbon
Keto form tautomerizes to…
Enol form
- Double bond between the two carbons with an alcohol group branching off the carbon
Guanine is usually found in what tautomeric form? what does it tautomerize to?
Usually found in keto form, tautomerizes into enol form
Adenine is usually found in what tautomeric form? What does it tautomerize to?
Usually found in amino form, tautomerizes into imino form
Thymine is usually found in what tautomeric form? What does it tautomerize to?
Found in keto form, tautomerizes into enol form
Cytosine is usually found in what tautomeric form? What form does it tautomerize to?
Found in amino form, tautomerizes into imino form
As bases tautomerize, what happens to the hydrogen donors and acceptors?
The hydrogen donors and acceptors change around
How are tautomeric shifts defined in the context of base pairing?
Tautomeric shifts are the spontaneous rearrangements of nitrogenous bases that allow for hydrogen bonding of mismatch base pairs
- Can lead to substitution mutations
Describe how DNA’s flexibility helps in DNA repair
- The sugar-phosphate backbone serves as a hinge to rotate the base
- Leads to homologous recombination and DNA repair
Describe the Mica experiment
- DNA is immobilized on a Mica surface (a flat, naturally occurring mineral)
- DNase I, a bulky enzyme, cleaves the phosphodiester bonds in the backbone. DNase I is too bulky, so it only cuts the DNA at sites where it’s most exposed (these sites occur at regular intervals)
- The fragments are separated on an agarose gel, where the fragment sizes seemed to be multiples of 10.5. This indicated that the enzyme would make a cut in the strand not attached to the mica every 10 nucleotides or so
What conclusions could be made by 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
Explain how the major groove is rich in chemical information
In the major groove, the different base pairs expose unique patterns of hydrogen bond donors (D), acceptors (A), and other chemical groups like methyl groups (M) and nonpolar hydrogen (H), which provide a “chemical signature” for each base pair that is more distinct than in the minor groove.
- the minor groove presents less distinctive features, making it harder to distinguish between base pairs.
- This method helped with DNA sequencing in the past (but was labouring)
Pattern from left to right of hydrogen donors, hydrogen bond acceptors, nonpolar hydrogens and methyl groups in major groove for A-T base pair
ADAM
- if A is on the left and T is on the right
Pattern from left to right of hydrogen donors, hydrogen bond acceptors, nonpolar hydrogens and methyl groups in minor groove for A-T base pair
AHA
- If A is on the left and T is on the right
Pattern from left to right of hydrogen donors, hydrogen bond acceptors, nonpolar hydrogens and methyl groups in major groove for G-C base pair
AADH
- If Guanine is on the left and Cytosine is on the right
Pattern from left to right of hydrogen donors, hydrogen bond acceptors, nonpolar hydrogens and methyl groups in minor groove for G-C base pair
ADA
Direction of helix orientation in B DNA
Right
Number of base pairs per turn in B DNA
10.5
Rotation per residue in B DNA
36 degrees
Length of A DNA (relative to normal B DNA)
Short (squeezed)
Number of base pairs per turn in A DNA
11
Rotation per residue in A DNA
33 degrees
Length of Z DNA (relative to normal B DNA)
Long
Number of base pairs per turn in Z DNA
12 (6 dimers)
- The helix is arranged in such a way that every two base pairs (a dimer, like CG or GC) form a repeating unit
Rotation per residue in Z DNA
30 degrees (60 degrees/dimer)
__ DNA and __ DNA are both right-handed, while __ DNA is left-handed
B, A, Z
Diffraction pattern lines are (parallel/perpendicular) to the actual lines in the structure
Perpendicular (90 degrees)
In photograph 51 (Rosalind Franklin’s X-ray diffraction), there was a missing 4th layer line. What was this a result of, and what did this show regarding DNA?
The missing 4th layer line was due to destructive interference, and showed that DNA was a DOUBLE helix (because the signals from both strands cancelled each other out)
- Also showed that DNA has major and minor grooves because not all signals had destructive interference (showed that DNA is asymmetrical)
What’s the pitch of a DNA molecule (the distance between similar structures)?
34 Å
- determined by layer lines on photograph 51
What’s the rise of a DNA molecule (distance between adjacent nucleotides)?
3.4 Å
- determined by radius of image of photograph 51
What does the distance between adjacent dots in the center of photograph 51 tell us?
The diameter of the DNA helix, which is 20 Å
- determined by radius between spots on photograph 51
What is denaturation and what two things usually cause it?
Denaturation is the disruption of hydrogen bonds that normally used to maintain the structure and function of a macromolecule
- Caused by heat or extreme pH
What is melting temperature (Tm)?
Tm is the transitional mid-temperature at which 50% of double-stranded DNA is denatured into single-stranded DNA.
What are the two factors that affect Tm?
- GC content: higher GC content= higher Tm (because GC has lower entropy, so it’s more stable and because it has an extra hydrogen bond)
- Ionic strength of solution: higher salt concentration= higher Tm (because the phosphate backbone of DNA is negatively charged and higher salt= more cations= decreased repelling force between the duplex = increased dsDNA stability = higher Tm)
How can we tell if DNA is denatured based on UV absorbance?
DNA absorbs UV at a wavelength of ~260 nm. ssDNA absorbs >40% of UV at 260 nm than the dsDNA (because bases are more exposed)
- Known as the hyperchromic effect
When dsDNA is heated, it is _____. If it is cooled after, it is _____
When dsDNA is heated, it is denatured. If it is cooled after, it is re-associated/re-natured/re-annealed
Describe DNA hybridization
ssDNA can re-associate with another strand that has “similar” sequence to form a hybrid dsDNA
What has DNA hybridization helped with in terms of sequencing in the past?
In the past, when sequencing technology was limited, DNA hybridization helped find new genes and diseases. It did this by finding the complement of disease genes.
What 3 experimental methods is hybridization especially important for?
- Southern blots (uses DNA:DNA hybrid) and Northern blot (uses DNA:RNA hybrid)
- DNA and RNA microarray
- Next-Generation Sequencing
What is DNA topology?
The mathematical study of the DNA properties in deformation, twisting and stretching
Prokaryotic DNA is also known as…
Covalently closed, circular DNA (cccDNA)
Describe eukaryotic DNA and prokaryotic DNA in terms of flexibility
Eukaryotic DNA: Linear, can freely rotate and unwind
Prokaryotic DNA: topologically constraint
- In linear DNA, the ends can unwind to relieve any torsional stress caused by twisting. However, in prokaryotes, where DNA is circular, there are no free ends to allow the molecule to untwist on its own. Any change in twist or supercoiling must be actively managed by enzymes because the closed structure keeps the strands constrained.
How does DNA become topologically constrained by replication?
When DNA becomes unwound for replication, there is torsional stress applied to the sides of the unwound part. More bulgier twists end up forming as the DNA twists more and more.
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
Describe the linking number (Lk) of DNA
- Describes the topology of a DNA molecule
- Tells us the amount of torsional stress put on a given DNA molecule
- Tells us how many turns it would take for the two strands of DNA to completely separate
- Is an integer
What are topoisomers?
Different forms of cccDNA molecules that differ only in their linking number
True or false: topoisomers migrate the same distance on an agarose gel
False
- When a cccDNA undergoes supercoiling and is resolved on an agarose gel, there is no single band but rather two or more bands
- Order of travel from least to most migration is open circular -> linear -> supercoiled (not many loops) -> supercoiled (many loops)
What is supercoiled DNA? What is supercoiling determined by?
Supercoiled DNA is DNA that twists upon itself because it is underwound or overwound (and thereby strained) relative to B-form DNA
- Supercoiling is determined by writhe, but not by twist -> writhe describes how the overall double-stranded DNA helix is coiled or looped in three-dimensional space (e.g., forming supercoils).
- While twist reflects local helical structure, it’s the writhe that determines whether the DNA becomes supercoiled
Positive supercoiling is also known as…
Overwound DNA
Negative supercoiling is also known as…
Underwound DNA
Positive supercoiling is when DNA twists…
In the same direction as the DNA
Negative supercoiling is when DNA twists…
In the opposite direction as the DNA
Most DNA is found in what type of supercoiling?
Negative supercoiling
Does negative or positive supercoiling require more energy to unwrap?
Positive
What type of organisms have positive supercoiling and why?
Some bacteria (thermophils) have positive supercoiling (live in a harsh environment, so they require more stable DNA since it’s harder to unwind and takes more energy)
What are topoisomerases?
Enzymes that create/relief supercoiled DNA by changing the topology of DNA
What numbered nitrogen forms a glycosidic bond with the sugar in nucleic acids in pyrimidines and purines?
N1 for pyrimidines, N9 for purines
Nucleosomes introduce ______ supercoiling, by introducing _______ nucleosomes
Nucleosomes introduce negative supercoiling, by introducing left-handed nucleosomes
How would a relaxed, circular DNA molecule go from relaxed to supercoiled with 4 writhes?
The DNA is broken by the topoisomerase, one helix is rotated 360 degrees four times, then the ends are rejoined.
What are 3 functions of supercoiling?
- To reduce the space and allow for DNA to be packaged into the small nucleus
- To prevent or resolve entanglement of the DNA during cell division (butterfly chromosome structure arises due to DNA condensation. Important to maintain the genome structure to prevent DNA breakage)
- Positive supercoiling protects DNA from thermal denaturation and regulates gene expression in extreme conditions (like in thermophil bacteria - but these organisms with positive supercoiling have decreased gene expression compared to negatively supercoiled organisms)
What is a disadvantage of positive supercoiling for replication and transcription?
Energy is needed to unwrap the DNA for these processes
How does negative supercoiling help strand separation?
Negative supercoiling stores the free energy required to facilitate strand separation
- If a negatively supercoiled plasmid is “forced” to have the relaxed conformation, it will leave a region unpaired. So, introducing negative supercoils is a way of favouring unwinding of the DNA.
Describe how supercoiling can be removed using DNase I?
- By mild digestion with DNase I
- “nick” the dsDNA by breaking a single phophodiester bond to allow for free rotation of the DNA around the other backbone
- This free rotation unwinds the supercoils, effectively relaxing the DNA and bringing it back to its normal, relaxed state. Think of it like cutting one string of a tightly twisted rope. When you cut it, the rope can unwind and lose the extra twists.
Type I topoisomerase (Topo I) is frequently just referred to as…
Topoisomerase
Topo I makes a (ssDNA/dsDNA) cut
ssDNA
Topo I (does/does not) require energy
does not
- Relaxes supercoils “passively”
How many writhes does topo I decrease a supercoiled piece of DNA by? What does this reduce the linking number by?
1 writhe
- Reduces the Lk by 1
What are the five steps involved in the reaction of topo I?
- DNA is nicked by the attack of tyrosine
- The other side of the cleaved DNA is “held” by the enzyme
- The uncleaved DNA strand passes through the opening. This relieves the torsional strain from supercoiling.
- Reverse of the nicking reaction (the 5’ phosphate group of the broken strand is re-ligated (rejoined) to the 3’ hydroxyl group on the other side of the break)
- Once the DNA strand is re-ligated, the now relaxed or untwisted DNA is released from the enzyme.
Why is energy not needed for topo I to act on DNA?
- topo I is covalently linked to the nicked DNA through the OH of its tyrosine (cuts through one of the phosphodiester linkages)
- At the start of the reaction, one phosphodiester bond is broken, whereas another one is formed while strands rejoin
- So the number of phosphate bonds is the same throughout the reaction, meaning that topoisomerase I is energetically neutral
True or false: type II topoisomerases (Topo II) are also known as gyrases
False; Topo II is frequently called “gyrase” but this is not strictly correct because some type II topoisomerases are not gyrases
What is gyrase? Where is gyrase found?
A type II isomerase that introduces negative supercoils; found in prokaryotes but not eukaryotes
Topo II makes a (ssDNA/dsDNA) cut
dsDNA
Topo II (does/does not) require energy
Does
How many writhes does the topo II reduce supercoiled DNA by? How much does topo II decrease the linkage number by?
2 writhes
- Decreases Lk by 2
What are gyrases important for?
Important in separating entangled DNA in the cell, e.g. after DNA replication
- DNA gyrase, a type of topoisomerase found in bacteria, introduces negative supercoils into DNA. This process helps alleviate the positive supercoiling that occurs ahead of the replication fork during DNA replication or transcription.
Can topoisomerase I catenate/decatenate two double stranded circular plasmids?
No, topo II can do this reaction though
- If there’s a strand of ssDNA on both plasmids then yes, topo I can catalyze these reactions
The greater the length of reaction between a DNA molecule and a topoisomerase…
(explain what you would see on a gel)
The DNA becomes more relaxed (moves from the lower, more supercoiled bands to the higher, more relaxed bands).
- Lk decreases and eventually, each of the adjacent topoisomers in each lane differ by 1 Lk.
What is Ethidium Bromide (EtBr) used for in the lab?
Staining DNA in agarose gel
What can ethidum bromide (EtBr) do to DNA?
EtBr decreases the rotation of DNA from 36 degrees to 10 degrees by changing the space between adjacent base pairs
If a T tautomerized into its enol form, what would it pair with?
G
If a C tautomerized into its imino form, what would it pair with?
A
What direction must the B DNA be twisted in order to get a positive supercoil?
Clockwise (in the direction of the DNA twist)
What direction must the B DNA be twisted in order to get a negative supercoil?
Counterclockwise (opposite to the direction of the DNA twist)
Which direction does a negative supercoil loop around a histone core?
Clockwise
What direction does a positive supercoil loop around a histone core?
Counterclockwise
What reactions is Topo II useful for?
Catenation/decatenation of dsDNA (after replication), and separating entangled DNA after replication
Why does π stacking exist?
Ensures that the bases are facing the inside of the helix
Which of the forms of DNA molecules (B, A or Z) is hollow on the inside?
A DNA
