DNA Structure and Supercoiling Flashcards
Lecture 1 (Complete) (Video notes included)
Why is Fred Griffith important?
In 1928 he demonstrated the Transforming Principle: material isolated from heat-killed virulent bacteria can transform non-virulent bacteria into a virulent form. He used Streptococcus pneumoniae.
Why were Oswald Avery, Colin MacLeod and Maclyn McCarty important?
In a follow-up experiment to Griffith’s Transforming Principle, in 1944 they demonstrated nucleic acids were the material that carried the information, by fractionating material isolated from heat-killed bacteria.
Transforming activity destroyed when nucleic acids treated with deoxyribonuclease but not with ribonuclease, so transforming principle was DNA.
Why were Alfred Hershey and Martha Chase important?
In 1952 they confirmed DNA as the genetic material by labelling bacteriophage T2 with either 35S [labels proteins] or 32P [labels nucleic acids].
Only 32P detected in infected bacteria and in phage progeny.
35S isolated in phage ghosts which fail to enter bacteria.
What is a polynucleotide?
Give two examples
A polymer of a nucleotide
DNA and RNA
When writing nucleotide sequences, which direction is convention?
5’ to 3’
How are nucleotides joined together in DNA?
A phosphodiester bond between the 3’ OH of one sugar and the phosphate attached to the 5’ hydroxyl of the next sugar.
What is the repeating unit of DNA?
The sugar phosphate backbone
What is a dAMP?
A nucleotide
What is the similarity / difference between a DNA sugar and RNA sugar?
Both have pentose sugars (5 carbon) but for RNA there is an extra oxygen at the 2’ carbon (OH) whereas for DNA it is only hydrogen (H) .
Therefore ribose is more reactive.
True or false:
Nitrogenous bases / nucleobases are planar rings, typically uncharged under physiological conditions.
True
True or false:
Nitrogenous bases / nucleobases are planar rings, typically charged under physiological conditions.
False:
Nitrogenous bases / nucleobases are planar rings, typically uncharged under physiological conditions.
What does it mean if a base is a pyrimidine?
It is made up of a single ring
What does it mean if a base is a purine?
It is made up of a double ring
What are single-ringed bases called?
Pyrimidines
What are double-ringed bases called?
Purines
Which bases are pyrimidines?
Cytosine, Thymine, Uracil
Which bases are purines?
Adenine, Guanine
True or False:
Positions in sugars are referred to using the prime symbol
True
True or False:
Positions in sugars are referred to without the prime symbol
False:
Positions in sugars are referred to using the prime symbol
True or False:
Positions in bases are referred to using the prime symbol
False:
Positions in bases are referred to without the prime symbol
True or False:
Positions in bases are referred to without the prime symbol
True
What is a tautomer?
A molecule (eg; a base) where a proton has migrated to a different place
What percentage of bases are in the tautomeric form at any one time?
<0.01%
(But still have a significant effect, as human genome is 3Gbp = 3000000000 base pairs. 0.0033% of this number is = 100,000 bases)
True or False:
Bases flip back and forth between the common form and rare tautomeric form.
True
True or False:
If a base transforms to the tautomeric form it cannot return to its original form.
False:
Bases flip back and forth between the common form and rare tautomeric form.
When does it matter if a base is in the common or tautomeric form?
During replication (can result in damage and mutations)
What is a nucleoside?
A base and a sugar, without the phosphate
How are nucleosides named?
They are named for their base and their sugar, eg; adenosine (ribose), deoxyadenosine (deoxyribose)
How are bases and sugars joined?
A glycosidic bond between the C1’ of the sugar and the:
N1 of a pyrimidine
or
N9 of a purine
Where do phosphates bind to nucleosides to create nucleotides?
On the C5’ of the sugar
What additional biological functions are nucleotide monomers involved in?
ATP , energy carrier and phosphoryl donor
CoA , acyl group activation and transfer
S-adenosyl methionine , methyl group donor
NAD+ /NADH NADP+ / NADPH , oxygen-reduction
FAD/FADH2 , oxygen-reduction
What are Chargaff’s rules?
Amount of purine bases = Amount of pyrimidine bases
so [A]+[G]=[C]+[T]
Amount of guanine = amount of cytosine
so [G]=[C]
Amount of adenine = amount of thymine
so [A]=[T]
What did Franklin and Wilkins do?
X-ray diffraction analysis of DNA showed:
Helical structure characteristic of two intertwined helices
Spacing of lines suggested dimensions of helix
What dimensions of the DNA helix were inferred from Franklin and Wilkins’ experiment?
3.4 nm, approximately 10 bp / turn [34 Å]
0.34 nm rise per bp [3.4 Å]
2 nm helix diameter [20 Å]
What are Watson-Crick base pairs?
A pairs with T with 2 H-bonds
C pairs with G with 3 H-bonds
A-T and C-G base pairs have similar widths
Two DNA strands associate via weak hydrogen bonds to form double-stranded DNA
What is the structure of B-DNA?
(helix diameter, bp/turn, distance between bases, morphology, major and minor grooves, helix location)
2 nm helix diameter
10.5 bp / turn
0.34 nm between bases
One full turn of helix is 3.57 nm
Long, thin morphology
Major groove: Wide, intermediate depth
Minor groove: Narrow, intermediate depth
Helix axis location: Through base pairs
Describe the properties of B-DNA
Two complementary, antiparallel, polynucleotide DNA strands wind around each other, forming a right-handed (clockwise) double helix.
Hydrophilic sugar phosphate backbone on outside of molecule, hydrophobic bases form a stack on the inside.
Van der Waals interactions between bases stabilise DNA.
Base stacking can contribute significantly to stability but depends on neighbouring bases.
What is the importance of the major and minor groove?
In the major groove:
-Each base pair provides different chemical information
- Sequence specific binding proteins can recognise the different sequence combinations of hydrogen bond acceptors, donors and methyl groups available
In the minor groove:
- A-T & T-A are indistinguishable from each other, as are G-C & C-G
Fill in the gaps:
Proteins that bind to DNA but are not sequence specific bind to the ______ groove.
Proteins that bind to sequence specific DNA bind the ______ groove.
Proteins that bind to DNA but are not sequence specific bind to the minor groove.
Proteins that bind to sequence specific DNA bind the major groove.
What conformation is DNA predominantly in in cells?
Why?
B conformation
High humidity
What conformation is DNA in when there is low humidity?
A DNA
What conformation is DNA in when there is high humidity?
B DNA
Diameter = 2 nm
Right-handed helix (clockwise)
10.5 bp / turn
What conformation is DNA in when exposed to lots of methylation of cytosine, torsional stress, high salt concentrations?
Z DNA
What are some non-B DNA structures formed in genomic repetitive sequences with their sequence requirements?
Cruciform conformations, require inverted repeats
Slipped (hairpin) structure conformations, require direct repeats
Quadruplex conformations, require oligo (G)n tracts
[AG3(T2AG3)3 sequence, Single Strand] [Hoogsteen base paring] Seen in telomeres
What is supercoiled DNA?
DNA under tension that twists in on itself
What is relaxed DNA?
Open, uncoiled circular DNA
Where can supercoils form?
In constrained linear DNA, during replication for example
How are supercoils generated?
If parts of the DNA within the helix are untwisted, the whole molecule will wrap around itself to compensate for the amount of twists missing
What is Lk?
Linking number:
Number of times one strand wraps around the other (fixed for circular DNA and constrained linear molecules)
What is Tw?
Twist
Number of turns in a DNA fragment (+1 per 360 twist)
What is Wr?
Writhe
Number of supercoils (can be positive or negative)
What equation involves linking number, twist and writhe?
Lk = Tw + Wr
How can you tell if something is positively or negatively supercoiled?
If you unwind the supercoil and this opens up / loosens the DNA strand (separates a the strands of a small section) this is a negative supercoil
What enzymes use energy to introduce and remove supercoils from DNA, by temporarily breaking DNA and twisting it?
Topoisomerases
What are topoisomerases?
They are enzymes that use energy to introduce and remove supercoils from DNA by temporarily breaking DNA and twisting it
Why is negative supercoiling useful?
It facilitates DNA strand separation, as unwinding negative supercoils opens up the DNA strand
Why is there a major and minor groove formed?
Because of hydrogen bonding, the opposite sugar-phosphate backbones are not equally spaced
Describe the structure of A DNA
(helix diameter, bp/turn, distance between bases, morphology, major and minor grooves, helix location and direction of turn)
Diameter = 2.6 nm
Right-handed helix (clockwise)
11 bp / turn
Even sized grooves
Short and fat morphology
Major groove: extremely narrow, deep
Minor groove: very wide and shallow
Helix axis: major groove
Can be induced by DNA binding proteins
Describe the structure of Z DNA
(helix diameter, bp/turn, distance between bases, morphology, major and minor grooves, helix location and direction of turn)
Diameter = 1.8 nm
Left-handed helix (anticlockwise)
12 bp / turn
Elongated and thin morphology
Major groove: flattened out on helix surface
Minor groove: extremely narrow, very deep
Alternating pyrimidine / purines
