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Why is the sugar called 2'-deoxyribose?
Because there is no deoxyribose at position 2'
Why is it necessary to denote positions on the ribose with primes?
To distinguish them from positions on bases
How is the nitrogenous base joined to 2'-deoxyribose?
Via a condensation reaction where a molecule of water is removed when the OH on the 1' carbon of the sugar and the N9 of the purines/N1 of the pyrimidines join together to form a glycosidic bond
What is a glycosidic bond?
A type of covalent bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate.
The sugar and base alone are called a?
How does the phosphate group bond to 2'-deoxyribose?
Via a condensation reaction where a water molecule is removed when the phosphate joins the OH on the 5' carbon on 2'-deoxyribose
What is the resulting compound called when the phosphate group bonds to 2'-deoxyribose?
A 5' phosphomonoester
Adding phosphate(s) to nucleotides creates?
How are mononucleotides joined to other nucleotides to form polynucleotides?
They are joined to each other via the 3' OH of 2'-deoxyribose of one nucleotide and the phosphate group (attached to the 5' OH) of another nucleotide
What kind of linkage is stated in the above card?
2 adjacent sugars are now linked via a what group?
A phosphoryl group
What is the significance of the phosphodiester linkage linking at the 5' and 3' OH groups?
It imparts an inherent polarity to the DNA chain, causing DNA chains to have a free 5' phosphate/OH at one end and a free 3' phosphate/OH at the other
In convention do we write DNA sequences from 5' to 3' or vice versa?
From 5' to 3' is correct
Do you know how atoms in purine rings are numbered, and how the purine bases A and G differ?
This is how. Note how the the numbering goes clockwise and anticlockwise, and note where the numbering start(ie: where 1 and 7 start)
Do you know how atoms in pyrimidine rings are numbered, and how the pyrimidine bases C and T differ?
This is how. As before, note where the numbering starts
Each of the bases exists in 2 states which are called what states?
do only selected bases have tautomeric states?
No, all of them have tautomeric states
Do the tautomeric states have fixed values or do they exist in equilibrium?
They exist in equilibrium
What is the position of the equilibrium
It means far to the side of the conventional structures
What does "conventional structures" refer to?
The amino and keto configurations
What is a keto group?
If so, what are the corresponding "unconventional" groups?
The imino group and the enol groups
What is an imino group?
A carbon-nitrogen double bond
What is an email group?
an alkene bonded to an OH group
How does an amino configuration convert into an imino configuration?
A H atom from the amine group moves to a nearby N atom to form a C=N-H
How does a keto configuration convert into the enol configuration?
A H atom from an N-H nearby moves to a carbonyl group, creating a C-O-H
In the previous card we saw guanine's 2 tautomeric forms. However, in enol form, the OH was not bonded to an alkene. How come
While in the diagram the double bond was seen to be from the C to the N, we have to remember that it is an aromatic compound, hence its pi electrons will be delocalised over the entire ring, giving all bonds a double bond character.
What is the significance of the DNA bases having the capacity to form alternative tautomers?
It is a frequent source of errors during DNA synthesis
The 2 strands in the DNA duplex are said to be arranged in a what fashion?
How is adenine able to H bond with Thymine? (what are the specific locations of the groups)
1. Between the exocyclic amino group at C6 on adenine and the carbonyl at C4 in thymine
2. at N1 of adenine and N3 of thymine, an amine group
How is guanine able to H bond with cytosine? (Specific location of groups)
1. Exocyclic NH2 at C2 on guanine with a carbonyl at C2 on cytosine
2. Between N1 of guanine and N3 of cytosine
3. Between the carbonyl at C6 of guanine and the exocyclic NH2 at C4 of cytosine
This specific base pairing results in a ____ relationship between the 2 DNA strands in a DNA duplex
Both AT and GC base pair have the same width; what is the significance of this?
It ensures that all 4 base pairs can be accommodated within the same arrangement without distortion of the overall DNA duplex; It ensures that different DNA base sequences will not affect the structure of the DNA molecule
At first glance, why does H bonding not appear to contribute significantly to the stability of the DNA duplex? (hint: aqueous
In single DNA strands, for every H bond formed with another DNA strand one would have to break a H bond with water, meaning the net energy decrease would seem insignificant
However, what has been overlooked that actually allows H bonding to contribute to the stability of the dna duplex?
When 2 DNA strands join via H bonds, the water molecules originally lined up on the bases will be displaced, creating disorder that increases entropy, hence stabilising the duplex (dG decreases)
Recap: what is another significant factor that helps stabilise the DNA duplex?
The base stacking interactions net been the bases, where electron cloud (pi) interactions contribute to the stability of the helix
More than the stability, what is another more important role of H bonds in DNA?
The base pairing specificity
Why can't A and C form H bonds?
Their acceptors and donors line up with each other, preventing H bonds from forming
Hence, why is AC unstable?
H bonds would have to be broken when the water molecules are stripped off the bases, but they would reform since A and C cant form bonds;
H bonds with water would be more favorable
The energetics favor the arrangement where bases are within the sugar phosphate backbone. However, what is an exception?
The phenomenon where bases protrude outwards from the duplex
What is this phenomenon called?
Why is base flipping important? (what processes require base flipping?)
1. Certain enzymes that methylate/remove damaged bases do so with the base in an extra-helical config, enabling the base to sit in the catalytic cleft
2. Enzymes involved in homologous recombination and DNA repair also do the same
Is base flipping energetically expensive?
Not really, because usually only one base it flipped at a time; if the all flipped at once, then it would be expensive
Is DNA a right handed or left handed helix?
(usually) right handed
(If you look from the transverse view)Each base pair is twisted from the previous one by about how many angles?
Hence, how many base pairs does it take to go completely around the helix? (to make one period)
About 10 since 36 x 10 = 360deg
Are the 2 grooves in the DNA duplex of equal size?
No, one is bigger than the other
The bigger and deeper groove is called the? Correspondingly, the smaller groove is called the?
Major and minor groove respectively
Why are the 2 grooves of unequal size?
Due to the geometry of the glycosidic bonds in opposite bases, the angle between 2 bonds is 120deg, resulting in the narrower angle generating a minor groove and the wider one a major groove
What would happen if the glycosidic bonds in opposite bases were 180deg to each other?
Then there would be no minor or major groove as they would be of equal size
Why is it said that the major and minor grooves contain chemical information?
The edges of each base pair are exposed in the grooves that create a pattern of H bond acceptors and donors that allow for identification of the base pair
Besides H bond acceptors and donors, what other kinds of groups allow for identification of base pairs?
Bulky hydrophobic groups(methyl groups) and hydrophobic hydrogens
How are the groups arranged in an GC base pair? (best to draw out)
How are the groups arranged in an AT base pair? (best to draw out)
Do the patterns allow for differentiation between AT and GC base pairs in both grooves?
Do the patterns allow for differentiation between base pairs and their reversed orders? (eg: AT and TA)
Only in the major groove, but not minor
Why is it that the minor groove does not allow us to distinguish between base pairs and their reverse orders?
Do the AT base pair the minor groove displays and AHA "code", which is read the same way forwards and backwards
Similarly in the GC base pair the minor groove has a code of ADA
Why is it said that the major groove is rich in chemical information while the minor groove is said to be less rich?
The major groove allows us to distinguish between many more different base pair configurations
Slight digression: What is mica?
What are phyllosilicates?
They form parallel sheets of silicate tetrahedra
What is an important property of mica that allows it to bind DNA?
Its surface is hydrophilic
Why is its surface hydrophilic?
Because all phyllosilicate materials are hydrated, having either water or hydroxyl groups attached to its surface
What part of DNA allows it to bind to the hydrophilic mica surface?
Its polar phosphate group
When DNA was bound to mica and treated with DNAse I, the enzyme could only cleave bonds in the backbone (top of DNA). Why?
Because the enzyme is bulky, hence, it faced steric difficulty reaching the bonds on the sides or bottom of the DNA due to the mica
Q: What bonds did the DNAase I cleave?
After treatment DNA fragments were separated via gel electrophoresis and fragments of 11, 21 and 32 bp were found. WSOT?
Since they were in multiples of 10.5, one can see that the number of bp per turn is ~10.5 (note that this value is for DNA absorbed on a surface; under physiological conditions the average in 10 per turn)
What are the 3 different forms of DNA?
A, B and Z
How does the A DNA differ structurally from B DNA?
The A form has 11 bp per turn compared to the B form's 10, and the A form has a much narrower and deeper major groove and broader and shallower minor groove
Under what conditions does DNA assume the B form?
Under high humidity
Under what conditions does DNA assume the A form?
Under low humidity
Do you expect the DNA to exist mostly in the a form or B form under physiological conditions?
Majority of DNA in cells is B DNA
When does DNA adopt the A form?
Usually during DNA-protein complexes
Even so, does DNA under physiological conditions match the structure of B DNA identically?
In what ways does physiological DNA differ from that of B DNA?
1. (as seen from the mica experiment) dna in solution is more twisted on average than B DNA (10.5 bp per turn vs 10)
2. B DNA is an average structure whereas real DNA is not perfectly regular
If DNA is not perfectly regular, what kind of variations does it have?
Two members of each base pair do not always lie exactly in the same plane, instead exhibiting a propeller twist where 2 flat bases counter rotate with respect to each other
If every base pair counter rotates, isn't the structure of DNA still regular in that sense?
The precise rotation per base pair is not constant. In particular, AT base pairs generally have a high degree of twisting
Why do bases exhibit this propeller twist?
They stack this way to exclude more water (presumably because when you slant 2 flat objects more and more the width of the gap between them narrows)
How does the propeller twist affect DNA structure overall?
1. Sometimes if can cause the entire duplex to bend in a certain direction
2. The width of the major and minor grooves is affected and hence their widths vary locally according to the degree of propeller twisting of various different base pairs
Hence, is DNA a perfect, regular double helix?
Can the N glycosidic bond at the 1' carbon of the ribose sugar be rotated?
If so, why is it that all DNA illustrations depict a conformation where the base is projected outwards from the sugar?
Because rotation about this bond is restricted and nucleotides usually assume the conformation described above
Why is rotation about the N glycosidic bond restricted?
Due to steric hindrance; it is more favorable for the bulky base to be far away from the sugar ring
There is a name for this conformation. What is the conformation described the previous 2 cards called?
The anti configuration
On the other hand, what is the conformation where the base is directly on top of the sugar called?
The syn configuration
In normal right handed DNA, what is the fundamental repeating unit?
A mononucleotide with an N glycosidic bond in the anti configuration
What is the fundamental repeating unit in left handed DNA?
A purine-pyrimidine dinucleotide
What is unique about the configuration of the N glycosidic bond in such purine-pyrimidine dinucleotides?
In the pyrimidine residues, the N glycosidic bond is in the anti config while in the purine residues it is in the sun config
Besides having a repeating purine-pyrimidine pattern, what kind of base composition especially promotes left handed DNA?
A deoxycytidine-deoxyguanosine repeating pattern. Ie:
In addition to this, what other factor encouraged formation of left handed DNA? (hint: has to do with ions)
High concentrations of cations
Why do cations promote formation of left handed DNA?
In left handed DNA, the phosphate groups are pushed together. Cations can help to shield negatively charged phosphate groups, greatly favouring this
Why is this left handed DNA called Z DNA?
Because of the alternating syn-anti configurations, the sugar phosphate backbone now appears zigzagged, giving it its name
Does Z DNA have a minor and major groove?
Kind of, they are of very similar width and depth
Why has Z DNA been difficult to study?
It is not a stable form of DNA and exists transiently only under certain cellular conditions
How is DNA denatured?
1. When it is heated above physiological temperatures (eg 100deg C) then thermal disruption will cause the 2 strands to come apart
2. Under conditions of high pH
Why does high pH denature DNA?
The pKa of guanine and thymine are roughly 9-10 pH, so if you reach/exceed that then the guanine and thymine residues will dissociate and form negatively charged N atoms on various places in the molecule, disrupting the H bonds and the stability
DNA can be analysed using UV spectrometry (~260 nm). Why?
The aromatic rings of the nitrogenous bases exhibit resonance, which allow DNA to absorb UV light
When DNA denatures, the absorbance of the solution containing DNA increases rapidly. This process is called?
What difference between ssDNA and dsDNA allows hyperchromicity to occur?
The ssDNA has higher absorbance than dsDNA
Why does ssDNA have higher absorbance than dsDNA?
The H bonds in dsDNA limit the resonance of the aromatic rings, presumable because electrons would crowd around H bond acceptors and away from donors. In ssDNA, this is absent, giving the electrons more freedom, increasing resonance of the rings
What does the rapid increase in absorbance tell us about the process of denaturation?
It is a very spontaneous process that occurs over a narrow and well defined temperature range
What about the renaturation process?
It is also very fast
What could be happening in the renaturation process that allows it to occur so quickly?
Once a complementary strand joins to another at a certain location, the remainder of the 2 strands will quickly "zipper up" and join together, at that point no longer separating again (unless temp increases)
Q: despite the rapid change in absorbance, it still happens over a range of temperatures. Which point is the melting point?
The midpoint of this transition
Is the melting point the same for all DNA?
Which factors affect the melting point of DNA?
1. GC content
2. the ionic strength of the solution
How does a higher GC content affect the melting point of DNA?
It will raise the melting point's temp
Why does high GC content raise the melting point?
I allows the DNA to be more stable
Why does high GC content cause the DNA content to be more stable?
1. Greater number of H bonds between GC base pairs
2. Base stacking interactions between GC base pairs and adjacent base pairs are more favorable
How does a solution with high ionic strength affect melting point affect melting point?
It raises melting point
Why does higher ionic strength raise melting point?
The cations shield the negative charge of phosphoryl groups, reducing the repulsion between phosphoryl groups on opposite strands, causing the DNA duplex to be more stable, increasing its melting point
Hold on, what if "high ionic strength" meant more anions? wouldn't this destabilise DNA?
Unless through electrolysis by placing only the cathode in solution (or some other method), most ionic solutions, especially in biological contexts, will have both cations and anions, so there will always be available cations to interact with DNA
Are all DNA molecules linear?
No, some are circular
Does linear DNA always remain linear?
In most cases, yes. However, in some special cases it can change according to environment (like the phage lambda genome that is linear when in the virus, but circularises via annealing of sticky ends when in E Coli)
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