Nucleic Acids Structure, supercoiling and degradation Flashcards
Readings: 305 - 316
There are 2 bonds between what base pair?
Adenine and thymine
There are 3 bonds between what base pair?
Guanine and cytosine
How many Angstroms (Å) is the length of one helical turn in DNA?
34
How many base pairs are in one helical turn of DNA?
10.5
What is the major groove between in DNA?
Helical turns
What is the minor groove in DNA structure?
Between strands
What is the width of DNA?
20 Å
What is the distance between each base pair?
3.4 Å
What two things stabilizes DNA double helix?
Hydrogen bonds and base stacking
When is A-DNA formed?
In high salt and low humidity situations
What is the helix sense for B-DNA, A-DNA and Z-DNA?
B-DNA: Right handed
A-DNA: RIght handed
Z-DNA: Left handed
What is the sugar pucker conformation for B-DNA, A-DNA and Z-DNA?
B-DNA: C-2’ endo
A-DNA: C-3’ endo
Z-DNA: C-2’ endo for pyrimidines
What is the glycosyl bond conformation for B-DNA, A-DNA and Z-DNA?
B-DNA: Anti
A-DNA: Anti
Z-DNA: Anti for pyrimidines and syn for purines
What is the structure of Z-DNA?
Sequence of alternating purines and pyrimidines, with Z-DNA binding proteins attached
What are the 3 conformations of DNA in order of most compact to least compact? (smallest grooves to biggest grooves)
A form
B form
Z form
What do B, A and Z DNA’s look looking ‘into’ them?
B: Center disk with spokes inside a circle
A: A circle within a circle
Z: A star in a circle
Describe a nucleoside with a syn glycosidic bond
The base points in the same direction as the pentose, that is, it is right over it.
Describe a nucleoside with a anti glycosidic bond
The base points away from the pentose ring
Which conformation of glycosidic bond is favoured in DNA? Why?
The anti conformation is favoured because the syn conformation has more steric hindrance due to crowding of the pentose ring
What is linking number (Lk)?
The number of helical turns in DNA
How can linear DNA form supercoils?
linear DNA between two anchored points can supercoil
What is LKo?
LK in relaxed DNA
How can LKo be determined?
By dividing the number of base pairs in strand by 10.5 (remember 10.5 bp per helical turn of B-DNA)
What is S for nucleic acids?
Number of supercoils in DNA
How can number of supercoils (S) be determined for DNA?
S = LK - LKo
What is ethidium bromide used for?
Used for visualizing DNA when exposed to UV, but also highly mutagenic. It intercalates in DNA like a base pair.
How does ethidium bromide effect supercoiling?
It decreases negative supercoiling
What is the LKo equation for ethidium bromide’s effect on DNA?
LKo = (# bp) / (10.5 + # of ethidium bromide)
Tou have a strand of circular DNA 200 helical turns (Lk = 200) and it is relaxed (Lko = 200). What does Lk become if DNA is negatively supercoiled by two helical turns?
Lk = 198 with a ΔLk of -2
How do positive supercoils affect the number of helical turns (Lk)?
They increase the number of helical turns
How do negative supercoils affect the number of helical turns (Lk)?
They decrease the number of helical turns
Does supercoiled DNA Lk decrease or increase further from a well in a western blot? Why?
Lk decreases further along a western blot, as DNA is more supercoiled further along a western blot.
What does topoisomerase I do?
Relaxes negative supercoils
What does topoisomerase II (DNA gyrase) do?
Introduces negative supercoils
What does Topoisomerase III do? Which class is it?
Specialized functions in DNA repair and replication. Type 1 topoisomerase
What does type I topoisomerase do?
Relaxes negative supercoils
What does type II topoisomerase do?
Introduces negative supercoils
What effect will type II topoisomerase have on the Lk of DNA?
Decrease Lk by introducing negative supercoils
What effect will type I topoisomerase have on the Lk of DNA?
Increase Lk by relaxing negative supercoils (will not relax above Lko)
How many helical turns does type I and topoisomerase I deal with in a single action? How many strands at once?
I turn per action along a single strand
How many helical turns does type II topoisomerases make in a single action?
2 turns per action.
What are nucleases?
Enzymes that catalyze the hydrolysis of phosphodiester bonds in nucleic acids
Name 6 varieties of nuclease
- ribonuclease (RNase)
- Deoxyribonuclease (DNase)
- Non specific nuclease
- Single strand nuclease
- Double strand nuclease
- Single and double strand nuclease
What are two modes of attack for nucleases?
- Endonuclease activity, hydrolyzes interior phosphodiester bonds
- Exonuclease activity, acts sequentially from one end to the other (ie. 5’ to 3’ or 3’ to 5’)
Give an example of two nucleases used in food digestion
Pancreatic DNase I and RNase A
how are nucleases used in gene regulation?
RNA turnover
What are the products of RNase phosphodiester bond cleavage?
A 5’ OH and 3’ Phosphate
What are the products of DNase phosphodiester bond cleavage?
A 3 OH and 5’ phosphate
What are three types of base specificity in nucleases?
- Specific for a single base (eg. RNase T1 specific of G)
- Specific for a particular class of base (eg. RNase S, specific for pyrimidines
True or false, alkalis hydrolyze both RNA and DNA
False, alkalis hydrolyze only RNA
however, Alkalis denature DNA
What configuration is double stranded RNA in?
A-DNA conformation
What are pucker conformations? List two
Pentose conformations (it’s not flat).
- C3’-endo, where 3’ carbon is protruding up
- C2’-endo, where 2’ carbon is protruding up
What is the norm for supercoils in nature?
Negative supercoils
Can loci in the genome ever have positive supercoils?
Yes, though temporarily
Why do thermophilic bacteria have positive supercoils?
Because it makes DNA more stable and able to withstand high temperatures
Which topoisomerase has a ‘controlled relaxation?’
Type I Topoisomerases
What is another name for topoisomerase II in prokaryotes?
DNA gyrase
Which type of topoisomerase requires ATP?
Type II, because it introduces negative supercoils, goes away from relaxed (lower energy) state
What type of topoisomerase can separate catenated chromosomes?
Type IV
What is the conventional direction of the top strand of DNA (when drawn)?
5’ to 3’
What phosphates are cyclic, what do they become susceptible to? What are results of this?
Susceptible to hydrolysis by water, making monophosphate intermediates (2’ and 3’)
Why can’t alkali solutions hydrolyze DNA?
Because it lacks the 2’ OH group that RNA has
What is topology with supercoiling?
A branch of mathematics that studies the properties of an object that do not change under continuous deformations.
Is most cellular DNA underwound or overwound?
Underwound, where the molecule has fewer helical turns than expected for B-DNA
If an 84 bp segment of circular DNA had one of its helical turns removed, how many base pairs would there be for every helical turn?
84 bp/10.5 bp per turn = 8 helical turns
84 bp/7 helical turns = 12 bp per turn
Explain why supercoiling occurs, thermodynamically
When there are more or less helical turns in a molecule of DNA, the molecule is more unstable and thermodynamically strained. The strain is accommodated by coiling the axis on itself.
How does topoisomerase change linking number at a molecular level?
It breaks one strand of DNA, rotates the end of it 360 degrees about the unbroken strand and rejoins the broken ends.
What is writhe (Wr)?
A measure of the coiling of the helical axis
What is twist (Tw)?
The local twisting or spatial relationship of neighbouring base pairs
What is the equation associated with Lk, Wr and Tw? Why?
Lk = Tw + Wr
Because when linking number changes, some strain is compensated with twist and some is compensated with writhe
What form of supercoiling is observed in isolated DNA?
plectonemic supercoiling, extended right hand coiling (like telephone knots)
What are two forms of compaction from supercoiling? Which compacts more and gives chromosomes their density?
- Plectonemic (most stable in solution)
- Solenoidal (eg. around nucleotides)
Solenoidal supercoiling compacts more. These forms are readily interconvertible
