Lecture 1 - DNA Structure and Supercoiling Flashcards
Name and describe the roll of some of the key figures in the search form genetic material.
1928 Fred Griffith – Streptococcus pneumoniae
* Demonstrated that material isolated from heat-killed virulent bacteria could transform non-virulent bacteria into a virulent form – ‘transforming principle’
1944 Oswald Avery, Colin MacLeod, Maclyn McCarty – follow up expt.
* Fractionated material isolated from heat-killed bacteria – demonstrated that nucleic acids were ‘transforming principle’.
* Transforming activity destroyed when nucleic acids treated with deoxyribonuclease (digests DNA), but not ribonuclease (digests RNA)
* Identified Griffith’s ‘transforming principle’ as DNA
1952 Alfred Hershey, Martha Chase (Necessary due to scepticism)
* Labelled bacteriophage T2 with either 35S (protein coat) or 32P (DNA)
* Only 32P detected in infected bacteria and in phage progeny
* 35S isolated in phage ‘ghosts’ that fail to enter bacteria
Confirms DNA as genetic material
What are Nucleic acids?
Nucleic acids store and transmit genetic information. DNA and RNA are polymers of nucleotides - polynucleotides.
Nucleotides are joined by a phosphodiester bond between the 3’ OH of one sugar and the phosphate attached to the 5’ hydroxyl of the next sugar. They are directional and the sequence is always written in the 5’-3’ direction.
Describe the composititon and structure of DNA and RNA.
Pentose sugars
DNA is made using deoxyribose as the sugar whereas RNA is made using ribose. The only difference between these sugars is a hydroxyl group on the 2’ carbon of the ribose sugar which makes it more reactive (has catalytic properties)
Nitrogenous bases
Bases are planar rings that are typically uncharged under physiological conditions
Pyrimidines: C, T, and U are attached to the sugar at position 1 via a glycosidic bond
Purines: A and G are attached to the sugar at position 9 via a glycosidic bond
Nucleosides and nucleotides
Nucleoside = Base + Sugar
Nucleotide = Base + Sugar + Phosphate
A base is joined to a sugar by as glycosidic bond between the C1’ of the sugar and the N1 of a pyrimidine or N9 of a purine (shorter word, larger structure)
What is base tautomerisation?
Bases can also exist as tautomer’s (a molecule that has a proton that has migrated to a different place). There at one time in the human genome are approximately 100000 bases that are tautomer’s and they have implications for the accuracy of DNA replication and therefore provide genetic variation.
E.g. If adenine is in the rare tautomer form a cytosine will form its base pair instead of thymine
Adenine and cytosine from imino groups in the tautomeric form whereas guanine and thymine form enol groups in the tautomeric form.
What are the roles of some nucleotide monomers?
Nucleotide monomers have diverse important functions in cells
Other than in DNA they have additional biological functions:
Adenosine triphosphate - energy carrier, phosphate donor
CoA - acetyl group activation and transfer
S-adenosyl methionine - methyl group donor
NAD+/NADH - oxygen reduction
How was the DNA double helix discovered?
- Chargaff’s base composition studies
Chargaff broke down DNA and calculated the proportions of bases and concluded
[A] + [G] = [C] + [T] - Elucidation of structure of DNA double helix (Franklin and Wilkins)
Key information came from X-ray diffraction studies of DNA fibres meant that they could conclude based on the diffraction pattern that DNA has a helical structure and also gave information of the distances between the base pairs etc. - Watson and Crick used model building to propose the structure of base pairs. They concluded that:
Two DNA strands associate via weak hydrogen bonds to form DS DNA. They concluded that A pairs with T via two hydrogen bonds and C pairs with G with three hydrogen bonds They concluded that the base pairs had similar widths.
Describe the structure of B DNA.
B-DNA consists of two complimentary antiparallel strands of DNA. The two polynucleotide strands wind around each other in a right-handed double helix (clockwise). The hydrophilic sugar phosphate backbone is on the outside of the molecule whilst the hydrophobic base pairs form a stack on the interior of the helix. Van der Waals interactions between the bases stabilize the interactions. Contribution of base stacking to stability depends on neighbouring bases.
Crystal structure of B DNA characterized in 1980 – confirmed key aspects of Watson and Crick’s model.
* Helix diameter is ~2 nm (1 nm = 10-9 m)
* 10.5 base pairs in one complete turn of helix
* Base pairs are 0.34 nm apart
* One full turn of helix is 3.57 nm (0.34 nm/bp x 10.5 bp/turn)
* In B-DNA, the helix forms a major groove and a minor groove - govern interactions with other molecules
* B-DNA is the predominant configuration in cells
What is the Major Groove in relation to DNA
The major groove is rich in chemical information. Each bp presents different chemical information in the major groove. Information is different for different sequence combinations which provides a basis for recognition by sequence specific binding proteins.
Different DNA sequences have different combinations of hydrogen bond acceptors, donors and methyl groups available
In the minor groove T-A and A-T base pairs and G-C and C-G base pairs present the same chemical groups and therefore can’t be distinguished.
Name some conformations of DNA.
B-DNA (high humidity) - Right-handed helix, 10.5 bp/turn, Predominant conformation in cells, Diameter 2nm
A-DNA (low humidity) - Right-handed helix, 11 bp/turn Even sized grooves. ‘A’ conformation can be induced by DNA binding proteins. Diameter 2.6nm.
Z-DNA (alternating pyrimidine/purines) - Left-handed helix, 12 bp/turn. Induced by methylation of cytosine, torsional stress, and high salt concentrations. Diameter 1.8nm.
Non B-DNA structures formed in genomic repetitive sequences
Cruciform - inverted repeats
Slipped (hairpin) structure - direct repeats
Quadruplex - AG3(T2AG3)3 single strand
Describe the topological variations found in DNA. (Supercoiling)
Open uncoiled circular DNA is said to be relaxed. Under tension it twists in on itself and is supercoiled. Supercoils can also form in constrained linear DNA
Introducing supercoils into DNA
To generate supercoiling, a circular molecule is cut and held at one end while the other end is twisted
When the two ends are reattached the DNA twists to restore the preferred number of bases per turn
This causes the DNA to wrap around itself in a coiled structure
Linking number (Lk) is the number of times one strand wraps around the other – for circular DNA and constrained linear molecules this is fixed.
Twist (Tw) is the number of turns in a DNA fragment (+1 per 360 twist)
Writhe (Wr) describes the number of supercoils and can be positive or negative
Lk = Tw + Wr
Many biological processes require DNA strand separation – this is facilitated by negative supercoiling
In negative supercoiling, unwinding the supercoils opens up the DNA strand
Topoisomerases introduce or remove supercoils from DNA in an energy-requiring process by temporarily breaking DNA and twisting it
