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If a linear DNA duplex originally had 5 twists and I add a 6th, can it twist back?
What if the DNA was not linear, but instead circular? Can it "twist back" from 6 twists to 5 twists?
Why could the linear DNA molecule twist back, but not the circular one?
Because the ends of the DNA strands in the linear DNA were free, allowing the 2 ends of the duplex to rotate independently
However, in the circular DNA, you can't untwist any one part without twisting another
even if you cant untwist the number of twists in a circular dna can you instead add twists?
No, as mentioned previously, twisting/untwisting the circular DNA locally will only lead to adjacent areas being untwisted/twisted, making the total number of twists unchanged
Hence, what can be said of the number of twists in circular DNA?
The number of twists in a circular dna is absolute and cannot be changed
Such covalently closed, circular DNA is said to be?
Is linear DNA free from topological constraints?
What kind of factors subject linear DNA to topological constraints?
1. Extreme length
2. Entrainment in chromatin
3. Interaction with other cellular components
How does extreme length topologically constrain DNA?
It prevents the bending of DNA (since to bend it you would have to bend the entire length/bend it elsewhere so other parts of the DNA are unaffected; which takes more energy than if it was just a short segment of DNA)
So, circular DNA can't be twisted? But doesn't DNA replication require the strands to separate, which requires untwisting?
Circular DNA can be twisted locally; it just can't increase/decrease the total number of twists
Even so, wouldn't continued untwisting in one area force the DNA to twist a lot in other areas, creating stress?
Yes it would
Then how are you able to replicate the entire DNA if you can only untwist so much?
You will need the use of topoisomerases to reduce the absolute number of twists to relieve stress
How do topoisomerases reduce number of twists in circular DNA if you just said the number of twists is absolute?
They break the DNA and rejoin it
What is the abbreviation for covalently closed, circular DNA?
In linear DNA, you can pull apart DNA strands without breaking the backbone. Is this possible for circular DNA?
What must occur in order to completely separate 2 strands of DNA in circular DNA?
Breaking of the covalent linkages in the sugar phosphate backbone must occur
The number of times a strand has to pass through the other to allow the 2 strands to separate is called the?
The abbreviation for linking number is?
The Lk is the sum of which two geometric components?
The twist and writhe
What are the abbreviation for twist and writhe?
Tw and Wr
What does the twist refer to?
The number of turns of one strand about the other; every period in the helix (one criss cross) is one twist (just imagine what you would get if you had 2 parallel strands and rotated one fully; the final shape is one twist)
Does the twist of DNA have a directionality/polarity?
Yes, because it is a right handed helix, therefore it can be said to be twisting in right handed manner
The right handed twisting of DNA is denoted as positive. Can DNA twisting be negative?
Yes, in Z DNA the helix becomes left handed
What is writhe?
It is the macroscopic twisting of multiple DNA helices about each other (whereas twisting refers to the twisting of a strand itself)
Writhing of DNA can take how many forms?
What are the 2 forms?
1. the interwound/plectonemic writhe
2. The toroid/spiral writhe
What are interwound writhes?
Where DNA helices "twist"/interwound about a long axis (which does not need to be straight)
What are toroid writhes?
Where the DNA is wrapped in a spiral fashion to form a "cylindrical" structure
What is the equation describing Lk, Tw and Wr?
Lk = Tw + Wr
What do you notice about the equation regarding the Wr term? (hint: it's about Wr having 2 forms)
Despite having 2 forms, there is only one term describing writhes
What does this tell you about interwound and toroid writhes?
They are topologically equivalent
Since they are topologically equivalent, what does this tell you about them?
They are interconvertible
How do you visualise the interconversion of interwound writhes to spiral writhes and vice versa?
If you pull that apart by pulling on the place marked X, then you get the second structure, which is unstable. You then form the 3rd, more stable structure by reforming 2 loops. The loops can then be stacked on each other to form a spiral
Can a circular DNA duplex have completely no writhes? Visualise an example
Yes, for eg a circular DNA flat on a plane
When there are no Wr, what is the Lk equivalent to?
The number of twists, Tw
Since for circular DNA the number of twists is absolute, what can be said about the Lk (in the previous case)
It is also absolute/it is invariant
(following previous example) Will the Lk change if the flat circular DNA molecule has writhes?
The Lk is an invariant topological property of the cccDNA
If that's the case, what would happen when Wr increases? Would the rule be broken and Lk increases?
No, the rule cannot be broken (unless the cccDNA is topologically changed). The Tw will decrease corresponding to the increase in Wr
If we imagine a cccDNA with a figure 8 writhe, how many times must each strand pass over the other to uncoil the writhe?
Once each, which adds up to 2 crossovers
Hence, when a cccDNA with a figure 8 writhe relaxes it's coil, how many twists are added to the cccDNA?
Therefore, one interwind of the DNA duplex is equivalent to how many twists?
Important Q: In one of the first cards, you said that the no of twists in the cccDNA is absolute, is this always true?
No, it can change depending on the number of writhes. That rule was just stated to explain the importance topological difference between linear DNA and cccDNA
The formation of writhes is known as?
Can writhes be referred to as supercoils then?
I guess you could, but usually supercoiling usually refers to the process of writhe formation, not the writhe itself
Given a cccDNA with Wr = 0 and having 10,500 base pairs, calculate the Tw. (hint: 10.5 bp make one helix)
By dividing 10,500 by 10.5, you would get 1000, which is the number of twists
What is Lk0?
It refers to the Lk of completely relaxed DNA, with no supercoils and in its "natural state" (of course, the 10.5bp twist still exists, so the Lk just equals the number of twists)
What is one way to relax supercoiled cccDNA?
By treating the DNA mildly with DNase I.
Wouldn't treating the DNA with DNase I simply just destroy the DNA?
Unless the DNAse is in exceedingly high concentration (which isn't the case, since we are only treating the DNA mildly) the DNAse will break an average of one/a few phosphodiester bonds per molecule
How does this help relax the DNA?
It allows the DNA to no longer be topologically constrained, allowing the broken strand to now pass over the unbroken strand essentially allowing the strands to rotate freely
Even so, the DNA is still broken in some parts. How do we join the DNA back together?
Another enzyme could be added to ligate the broken strands
Even so, not all the resultant DNA relaxed through this method will have an Lk exactly equal to Lk0. Why?
Because of the random rotational fluctuations that cause some DNA strands to be located while in a slightly supercoiled state
Random Q: can Lk in cccDNA assume nonintegral values?
No, Lk is always an integer
Follow up to random Q: why is Lk always an integer?
(my own exp) because there can be no such thing as a fraction of a helix in a circular DNA molecule, therefore in a relaxed state DNA always has integral Lk since it has integral Tw. Hence, even with Wr, the Lk will not change and stay an integer
How is the extent of supercoiling measured?
Extent of supercoiling: dLk
therefore: dLk = Lk - Lk0
Hold on, didn't you say Lk is invariant? Then how does two different values of Lk, Lk and Lk0, arise in the prev equation?
While they may be describing a cccDNA that has the same base sequence and composition, Lk0 refers to its theoretical most relaxed state, while Lk refers to astate which has the same Tw as Lk0 but with Wr. (same molecule, diff topological states)
random Q: if Lk is just Lk0 with Wr, why doesn't the Lk molecule just uncoil into the Lk0 state then?
Short ans: The Lk state is thermodynamically more stable
Long: Since Lk is invariant, an "uncoiling" (without breaking the backbone) would cause Tw to increase, which may not be favorable
if dLk is a negative value, then the DNA molecule is said to be?
If dLk is positive, then the DNA molecule is said to be?
Why is it unfair to compare the dLk of different DNA molecules(of diff lengths) directly?
Given 2 DNA molecules supercoiled to the same extent, A longer DNA molecule would be able to form more writhes, so it's dLk will naturally be higher than that of the shorter one.
Instead, what is a better and more fair way to compare the extent of supercoiling of different DNA molecules?
(Since Lk0 is proportional to the length of the DNA molecule)
what is the name for the term dLk/Lk0?
The superhelical density, assigned the symbol sigma
Is Wr negative when the DNA is negatively or positively supercoiled?
Hence, why is it that cells usually contain negatively supercoiled DNA?
Since Lk=Tw+Wr, if Wr is negative, then negative supercoils can be seen to be converted into untwisting of DNA, which aids in processes requiring strand separation
ie: negative supercoils can be seen as stores of free energy that can untwist DNA
In contrast, some thermophiles contain positively supercoiled DNA. Why?
Positive supercoils can be converted into more twisting of the DNA, which will cause them to be harder to separate, a desirable feature when living in especially hot environments
Important Q: in order to create a right handed helix, one would have the rotate the top end of the helix in what direction?
In an anticlockwise direction (to the left, not right!); just imagine trying to create a right handed helix with 2 parallel rubber bands
Hence, to make DNA more twisted, one would have to twist it in ehixh direction?
The anticlockwise direction (note that when I say anticlockwise, I am referring to rotating the top end, and looking at it from a top view)
Another important Q: when negative supercoils form (not uncoil) does DNA become more or less twisted?
It becomes more twisted
But why?! didn't you say negative supercoils could be converted to untwisting of DNA?
Because in order for it to be subsequently converted to untwisting, during its formation it has to initially lead to twisting
Hence, when imagining how negative supercoils are formed, we have to imagine how to further twist DNA
When a cccDNA forms an interwound, figure 8 writhe, at which point in the DNA molecule is if twisted the most?
Where the red circle is
When the cccDNA is rotated anticlockwise (once again, from top view) what direction does the DNA at the marked points rotate?
It rotates anticlockwise. Just imagine your fingers; join 2 fingers from each hand and rotate one hand anticlockwise; you will realise that at the fingertips the fingers counter rotate anticlockwise
Hence, what direction must cccDNA rotate to form negative interwound supercoils?
What if you wanted to create a negatively supercoiled spiral writhes? which direction would you have to rotate the DNA? Why
Anticlockwise. As mentioned previously, rotating DNA anticlockwise will make it more twisted, which is what we want when forming negatively supercoiled DNA (so we can untwist the DNA later)
For negatively supercoiled interwound DNA, is the helix formed from interwoven DNA left or right handed? Why?
Right handed. It is not difficult to see why; in your minds eye, imagine twisting a circular rubber band anticlockwise. It will form a right handed helix
When the DNA is rotated anticlockwise to form negatively supercoiled spiral writhes, are the spirals left or right handed?
They are LEFT HANDED. Just look at the diagram. If you still cant imagine it, emulator the diagram using 2 of your fingers, rotating one anticlockwise, then collapsing them to get the left handed spiral
Hence, would you expect DNA to be wrapped around nucleosomes in a left handed or right handed spiral?
What class of enzymes is able to change the "invariable" Lk of DNA?
What are topoisomers?
They are DNA molecules of the same length but with different linking numbers
Briefly speaking, (no need to describe mechanism) how do topoisomerases change linking number?
They relax supercoiled DNA
What if a cccDNA has no supercoils but is very twisted? Can topoisomerase act on it then?
Typically if a DNA molecule has too many twists it will naturally supercoil itself to achieve the ideal 10.5 bp per turn value,
ie: the scenario described above isn't legit
How many general types of topoisomerase are there?
What are their names?
Type I topoisomerase and type II topoisomerase
Briefly speaking, what are the main differences between type I and type II topoisomerases?
1. Type II topoisomerases make transient double stranded breaks in DNA, while type I topoisomerases only make one
2. Type II topoisomerases require ATP, but type I topoisomerases don't
What do you mean type II topoisomerases make transient double stranded breaks? Could you elaborate?
They make a double stranded break, then pass the broken strands through the unbroken strands, then reseal the break
What about type I topoisomerases? How do single stranded breaks relax supercoiled DNA?
Instead of passing 2 strands over the other 2, type I topoisomerases pass one strand over the other to sort of add/remove twists to remove writhes
It was said that topoisomerases can relax supercoiled DNA by changing the Lk. Can they increase the Lk this way?
Yes. When relaxing negative supercoils, the Lk will increase, since Lk=Tw+Wr, if Wr was negative and subsequently became 0, then Lk would increase
Do all topoisomerases relax supercoiled DNA?
No, there is one exception called DNA gyrase
What does DNA gyrase do?
It introduces, rather than relaxes, negative supercoils
Does DNA gyrase belong to either type I or II topoisomerases?
Yes, it is considered to be a type II topoisomerase
In what kind of cells can DNA gyrase be found?
What is the purpose of DNA gyrase?
It is responsible for the negative supercoiling of prokaryotic chromosomes, which facilitates the unwinding of DNA during transcription and replication
Why don't eukaryotes have DNA gyrase then?
(my own opinion) Eukaryotic DNA is linear, hence adding negative supercoils is not sustainable as it is able to uncoil itself
What does it mean for DNA to catenated?
it refers to when 2 circular DNA molecules are linked together like a chain
Then what does decatenation mean?
It refers to the process where catenated DNA is separated
Hence, besides relaxing supercoiled DNA, what other role do topoisomerases have in cells?
Catenating and decatenating dna
How could catenated DNA arise?
During the replicate of DNA, two molecules get entangled and catenated
Is linear DNA free from this problem?
No. While linear DNA cannot be catenated since they have free ends, due to their extreme length they can get tangled, making them hard to separate
Would you expect decatenation/catenation to be carried out by type I or II topoisomerases?
Decatenation/catenation of 2 DNA molecule requires 2 strands of one to pass over the other, making this a job for type II topoisomerases
In what cases can type I topoisomerases fulfill the role of decatenation/catenation
When there has been a nick or a gap in either DNA
Besides the above-mentioned cases of catenation and linear DNA entangling, what is another case which requires topoisomerases
When DNA becomes knotted
Why is it important for cells to be able to decatenate, untangle, and untie DNA?
It is important as it allows the 2 DNA molecules to separate into 2 separate daughter cells
How do topoisomerases break DNA?
The active site of the topoisomerase contains a tyrosine residue that attacks a phosphodiester bond, which causes the DNA to break, with one broken stand connected to the tyrosine via a phospho-tyrosine bond. The other strand terminates with an OH
Do topoisomerases require ATP to drive the reaction described above? Why?
No. The phospho-tyrosine linkage conserves the G in the phosphodiester bond (since both are ester bonds), hence, the reaction is able to proceed without the need to couple it to the high energy released of ATP hydrolysis
Then why is it that type II topoisomerases require ATP?
They require ATP not for the breaking of DNA, but for promoting conformational changes
How are the broken strands joined back?
Since the energy of the phosphodiester bond is conserved in the phospho-tyrosine bond, the DNA can be resealed by reversing the original rxn; the OH group in one of the broken ends attacks the phospho-tyrosine bond to form the phosphodiester bond
In order for the topoisomerase carry out its purpose of relaxing DNA, what must occur between DNA cleavage and rejoining?
The passage of a second strand/strands through the break
(The following cards will use topoisomerase I mechanism as an model to study how topoisomerases relax DNA)
The process whereby topoisomerase relaxes supercoiled DNA is called?
The relaxation cycle
To initiate the cycle, what is the very first step?
For topoisomerase to bind to a segment of melted DNA
Important Q: what is melted DNA?
It is DNA whose 2 strands have separated
Why would there just be random segments of melted DNA?
Remember in most cells DNA is negatively supercoiled, making it favorable for the strands to separate
After binding to melted DNA, what happens?
One of the strands then binds in a catalytic cleft in the enzyme, which contains the tyrosine intermediate. This will cause the DNA to be cleaved via the mechanism mentioned earlier
Q: if one broken end is bonded to the topoisomerase via the phospho-tyrosine bond, where is the other end?
It is also tightly bound by the enzyme
Why is it important for the other end to also be bound tightly by the enzyme?
So later on the enzyme is able to bring back the two ends to allow them to join back together; if the broken end not directly bonded to the topoisomerase is allowed to float freely then subsequently the bond it will no be easy to reform the DNA
What happens after cleavage of the DNA?
The topoisomerase undergoes a large conformational change to open up a gap in the cleaved strand
Why is it important to open up a gap in the cleaved strand?
To allow the second uncleaved DNA strand to pass through
After the strand passes T, what happens to the unbroken strand?
The unbroken strand binds to a donut shaped hole in the topoisomerase
After this another conformational change occurs. What happens during this conformational change?
The 2 ends of the broken DNA strand are brought back together and the strand is rejoined
One change occurs. What happens during this?
The DNA molecule is released
What is the key concept in this process that can be applied to type II topoisomerases?
The use of a "protein bridge" to hold the two broken ends of DNA together
What is the difference between the type I and II topoisomerases?
Type II topoisomerases are usually polymeric (dimeric or even tetrameric); there are usually 2 type II topoisomerase subunits that come together (to provide more than one active site tyrosine residues) to cleave DNA
Can DNA topoisomers be separated via gel electrophoresis (even if topoisomers are of the same length?)
What is the basis of separation of DNA topoisomers via gel electrophoresis?
The greater the writhe of the DNA, the more compact it's shape will be, and the more easily and further it will migrate through the gel matrix (which is agarose in most cases)
How sensitive is electrophoretic mobility to the topological state (Lk) of DNA?
Very sensitive. If you realise, in the previous diagram the separated DNA only differed from each other by Lk=1
fully relaxed DNA of bp 3990, 4011 and 3995 were separated electrophoretically. Why did the 3995 DNA migrate the furthest?
Since they are fully relaxed, their Lk should =Lk0. Since 3990 and 4011 are divisible by 10.5 their Lk=Lk0 so dLk=0. But 3995 is not divisible by 10.5, (Lk must be integer) so its dLk is slightly >0, making it have ~0.5 Wr, hence it migrate further
What could be concluded from the experiment described above?
Since DNA that differ in length by 21 bp has same mobility but DNA that differs by 5 has different mobility, that means that DNA has a helical periodicity of ~10.5 bp per turn
Why does ethidium cause DNA to unwind? (hint: it is a flat, multi ringed cation)
It's shape allows Ito to intercalate between the nitrogenous bases in DNA. It's positive charge shields negative charge of phosphoryl groups, allowing them to come closer, causing DNA to unwind
Ethidium fluoresces when exposed to UV rays. However, its fluorescence increases after exposure to DNA. Why?
(my own opinion) When exposed to DNA it intercalates between the base pairs, allowing it to participate in base stacking interactions that increase its resonance
Hence, what is ethidium usually used as?
A stain to visualise DNA
When an ethidium ion intercalates between bp, it causes DNA to unwind by how many degrees?
What happens when ethidium intercalates in cccDNA?
Since Lk for cccDNA is invariant, this will cause the Wr of the cccDNA to increase due to reduction in Tw
Hence, what will happen when ethidium is added to negatively supercoiled DNA?
It will lose its writhe and will relax
How does ethidium affect migration of DNA in gel electrophoresis?
It will cause negatively supercoiled DNA to migrate slower, and positively supercoiled/relaxed DNA to migrate faster
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