Topic 3 Flashcards
What is a nucleoside
What is a nucleotide
Sugar and base
Sugar base and phosphate
Why is dna called deoxyribose
No oh on 2’ carbon
Just H
What is a phosphoester bond
The bond between the phosphoric acid and the 5’ oxygen of the sugar
Just phosphoester, if whole dna it is phosphodiester
What is the glycosidic bond between in dna
The 1’ carbons OH and the nh of the base
Purines
Pyrimidines
Adenine guanine
Cytosine thymine
Recongnicze AGCT structures
Slide 6
Double helix orientation of strands
Antiparallel
Phosphodiester bond is how many linkages
Phosphoester
2
1
What method helps us to see the dna size
Agarose Gel electrophoresis, smaller further, larger slower
How many h bonds are between A-T
G-c
2
3
What are the two dna chains stabilized by
Perpendicular Base pairing and stacking
What causes the GC bases to be stronger binded together
The benzene ring like structures in the g and c bases causes the hydrophobic force to be greater and pushes the bases close together
Recording slide 10
What causes pi stacking
The hydrophobic effect dues to vanderwalls forces
More in red slide 10
Is dna right handed or left handed
Right handed
If base faces in toward the sugar its
If our away from sugar
What type of twistings do these cause
Syn (left hand twisting)
Anti (right hand twisting)
What determines the orientation of bases (anti or syn) in the dna
The glycosidic bind that’s connecting them to the sugar
What are isomers
Same formula different structure
Give examples of two isomers
Amide to imidic acid (the h on the N goes to o)
Enol to keto ( oh on enols goes to ch)
What are tautomers
Isomers that are interconverted by migration of a hydrogen atom
Give an example of how cytosine becomes a tautomer and how the h bond donors and acceptors in it change
The nh2 amino (was a donor) loses H and now becomes acceptor
Slide 12
Give an example of how guanine becomes a tautomer and how the h bond donors and acceptors in it change
The nh gives it h to c=O
Not the c=o that was acceptor becomes c-oh donor
Slide 12
What can cause misplacing between bases
Slide 13
What are tautomeric shifts
Random rearrangements of the nitrogenous bases (due to tautomerization)
lets h binding happen between base pairs that don’t usually pair together (mismatched)
What happens after dna replication if a tautomeric shift causes GT and not Gc pairing
Recording slide 14
What is an example of how DNA is flexible
Base flipping
What is an example of how DNA is flexible
Base flipping
What causes base flipping
The enzymes involved in homologous recombination and dna repair
What is base flipping
The enzyme cuts out the h bond in the mispaired base and flips it out
The phosphate backbone acts as a hinge that lets the base flip out of the dna
What is the MICA experiment used for
It determines helical periodicity
What is the steps of the mica experiment
Immobllilize dna on the mica surface
do a restriction digest with DNase 1 (cleaves the phosphodiester bond in the back bone)
Run it on a gel electrophoresis and get separated fragments of the dna.
DNase 1 is too bulky to cut down to the bottom strand of the dna that’s right on the mica. So only cuts the outside strand
What did the mica experiment tell us and how slide 17
There are 10.5 bp per turn of the helix , because Found that the smaller fragments are 10-11 nucleotides
The stronger signals are around 31 to 32, means more abundant dna there,
Multiple of 10.5 nucleotides in school band size meaning in each turn of dna there must be 10.5 nucleotides
Each bp is twisted 36 degrees from the previous one
What is the major groove
Deeper and wider than the minor groove
What do the major and minor grooves tell us
Can see what types of base pairing occurs in the bands based on the h bond donor and acceptors
Can see what nucleotide is on which stand of the dna (if gc or cg)
What form are most double helical dna in
The B from
What forms of dna are there
B Z A
What type of handedness does z form dna have, what does it look like
Left handed, longer than normal
What type of handedness does A form dna have, what does it look like
Right, shorter (squeezed) than normal
How do we know that dna is mainly B form
Through x ray diffraction where dna is in a crystal and an x ray hits it
The rays are diffracted through the crystal
Diffraction parent lines are
Perpendicular to the actual lines
What does photograph 51 tell us
Slide 27
The photo is a diffraction pattern of the dna helix
The space between the layers/mark the rise per base pair (3.4Angstroms)
The centre of the photo to the top show the pitch (the distance that the helix repeats itself, 34A)
Can tell there are 10 bp/turn by dividing 34 by 3.4 (P/R)
The fourth layer line missing tells us that the lines are assymetically place, that there are major and minor grooves in the dna, and helix is double stranded
The radius of the dna is 10A
What are the two strand of dna held together by
Hydrogen bonds
What is denaturation of dna
Disrupting the h bonds in the dna
What denatured dna
Heat or extreme pH
What is the melting temp (Tm) of dna
The transition temperature where 50% of dna is denatured to ss dna and 50% is still ds
What affects the meting temp of dna
- gc content
- ionic strength of the solution
How does GC content contribute to the melting temp
Other than the h binds what causes gc to be hard to break
If there are more GC , the melting temp increases
because the 3 h binds are harder to break apart , also GC has lower entropy (so it’s more stable and harder to break)
How does ionic strength contribute to the melting temp
More salt (ions), higher melting point
Since salt is cations, they stabilize the negative dna backbone
Stabilizing the backbone decreases the repelling force between the duplex (two strands), so increased dsdna stability and higher Tm
What wavelength does dna absorb
260nm
How can we tell if DNA is denatured?
Why
Look at the absorbance Because ssDNA absorbs >40% of UV than dsDNA (at 260nm)
This is because the base are exposed in ssdna and are absorbing
What do you do to reanneal dna
Make them cold
What is dna hybridizations
Using the denature and reanneal process to make ssDNA reanneal with another strand that has a “similar” sequence
This makes Hybrid dsDNA
How can hybridization be useful
Southern blots, northern blots
DNA and rna microarray (to assess mutations or indels)
Next generation sequencing
When looking at hybrids on a southern blot, what is the probe and what is the sample we want to detect
What about northern
The probe is DNA and the sample is also dna
The probe is dna but the sample is rna
What is cccDNA
Give an example
Covalently closed circular dna
The sugar phosphate backbones of this dna is covalently linked (to make it a circle)
Ex. A bacterial plasmid or very long dsDNA
What causes dna to be topologically constrained
Unwinding of the dsDNA during replication or transcription causes torsional strain and over winding at the ends
The bubble opens but the ends become twisted
What is the linking number
A integer number that represents the number of times it takes for one strand of dna to pass through the other strand for the 2 strand to be separated
How many turns are needed to unwrap the double helices so the whole thing is separated
Lk =
Twist (two stands passing through each other) + writhe (entire thing twisting in itself)
What are topoisomers
The topology of the DNA is different (but same dna strand)
They only differ in their linking number
How can we tell that a dna has topoisomers
When the cccdna is pit in a gel , we see two bands or more
These bands are the different topisomers
order from larger to smallest the topoisomers of cccdna
Explain why it’s like this
Open circular > linear > supercoiled (1) > supercoild (2, more coil)
The supercoild is more packed than the open circular to travels further
What is supercooling determined by
Writhe not twist
What is supercooling determined by
Writhe not twist
What is supercoiling
The circular dna twist upon itself because it’s underwound or overwound relative to its regular B form dna
When is something positive supercoiled
Negative
When it’s overwound
When it’s under wound
Which type of supercoiling requires less energy to unwrap
Underwound dna (negative supercoiling)
Most organisms have
Negative supercoiling
Most organisms have
Negative supercoiling
What kind of some organisms have + supercoiling and why
Animals that need to survive in harsh conditions, they would need more energy to unwrap the dna
Slide 45
What enzyme can create or relieve supercoiled dna
Topoisomerase
They change the topology of the dna
What type of super coiling can nucleosomes introfuce
Generally negative but also sometimes positive
How many times does dna wrap around the nucleosome
2 times and can start wrapping from either top or bottom
What way does the dna have to start wrapping on the nucleosome to be negative supercoild
What about postive
Comes from bottom
Comes from top
Why does supercoiling happen
To reduce the space the dna takes up and let it be packaged tightly into the small nucleus
Making it compact in chromosomes will prevent the dna strands from tangling during chromosomal segregation (to allow segregation to happen smoothly)
What does positive supercoiling help with
Protects the dna from thermal denaturation and regulates the expression of genes in extreme conditions
So since the postive supercoiling is harder to unwrap, less likely to be expressed, which save energy for other thing for the organism to survive
Slide 49
What does negative supercoiling help with
It stores the free energy needed for the circular dna strands to separate during transcription and replication
If the dna was relaxed, a region would be left unpaired , if negative supercoil the dna will unwinds easier
Slide 50
How can we remove supercoiling
Digest the dna with DNase 1
It nicks the dsDNA strand by breaking only one phosphodiester bond on one strand
This allows free rotation of the dna around the still attached backbone
Then this can relax the dna or twist it and make the supercoil
What are the two types of topoisomerase
Type 1 and type II
What is Type 1 topoisomerase
Just called topoisomerase
Makes a ssDNA cut (only one strand)
Passively relaxed supercoils (meaning it doesn’t need atp)
How do you count Lk from a supercoil diagram
Look at the knee of times the dna crosses itself
That’s the Lk
One nick of topoisomerase reduces the linking number by
1
Explain the reaction of topo 1
Topo 1 has tyrosine residues, these residues interact with the dna strands
The tyrosine cleaves one phosphoester linkage in singles strand of dna (via its OH group, it attaches to the cut end of the dna now)
The non cleaved strand is held by one part of topo 1 and the cleaved is held by the other (two hands)
The uncleaved strand gets pulled through the opening and the nicked strand it put back together
A loop is formed and dna is released
Why does topo 1 not need to use energy
At the start of the reaction, one phosphoester bond is broken and another is formed at the end to being them together
So the number of phosphate bind I. The reaction is the same so net 0 atp used
What is type 2 topoisomerase
Better way to call it is topo II but some call is gyrase
Makes a dsDNA cut (diff from topo 1)
Require ATP (diff from topo 1)
What is a gyrase
What is it in
A type II topoisomerase that introduces supercoils
In prokaryotes but not eukaryotes
What does topo II do
Unwinds the dna better
can fully separate two plasmids (topo 1 can’t because it only reduced Lk by one and plasmids has two)
Cuts linking number by 2 (topo I does only one)
Slide 58
When can topo 1 fully separate two plasmids
When on of the plasmids have one ssDNA
What are topo II important for in terms of dna replication
They can fully separate entangled dna in the cell after they’ve replicated
To two separate strands
On a gel with supercoild and relaxed dna reacting with topoisomerase, why do the bands blur as more reaction time passes
The topoisomerase is forming multiple intermediate forms of the dna which is why many diff sized bands show
In the gel with dna topo I and the different topoisomer bands if one band is higher than the other what is the difference in their linking number
The difference in linking number is 1
What is ethidium bromide used for
Dye used To visualize the DNA in PCr
But it’s a carcinogen
What is does ethidium bromide do to the dna
It inserts itself in between stack bases to make the dna seem longer that is actually is
Changes the degree of rotation from 36 to 10 (lower rotation, longer dna)
This means is decreases the amount of twist and changes the topology of dna
Blots:
SNOW
DROP
southern : dna
Why is gc stronger than at (the main reasons)
The hydrophobic force due to the benzene rings in g and c is higher than the force in at
The gc pi stacking allows stronger interaction between them
What makes the bases face inside the dna
The hydrophobic effect , they aggregates to stay away from water.
What nitrogen of ag form the glycosidic bind
What about CT
N9
N1
What did photo 51 tell you very important
DNA has major and minor grooves
Negative supercoil on nucleosomes is
Positive is
Left handed wrapping (starts from bottom)
Right handed wrapping (starts from top
What amino acids are in keto form
In amino form
Are these the dominant froms
GT
AC
