L1: DNA Structure and Supercoiling

Question 1

Experiments leading to elucidation of DNA as genetic material

Answer 1

1. Griffith; ‘Transforming Principle’ from heat-killed virulent bacteria which could transform non-virulent into virulent
2. Avery et al.; Fractionated material and demonstrated it to be nucleic acids. Broke down and lost transforming properties with deoxyribonuclease but not ribonuclease therefore DNA.
3. Hershey, Chase; Labelled proteins with 35S and nucleic acids with 32P - only 32P detected in infected bacteria and in phage progeny - doubly confirmed it as DNA

Question 2

Basic DNA structure

Answer 2

• Polynucleotides
• Top strand written 5’ to 3’
• Joined by a phosphodiester bond between 3’ OH of one sugar and the phosphate attached to the 5’ hydroxyl of the next

Question 3

Base Tautomerism

Answer 3

Typically, <0.01% of genome take imino tautomer. Implications for accuracy of DNA replication -> genetic variation.

Question 4

Nucleosides (Composition, naming conventions, dA vs dAMP)

Answer 4

Base plus sugar, named for base e.g adenosine, deoxyadenosine (dA). Adding phosphate group = nucleotide e.g. Deoxyadenosine monophosphate (dAMP)

Question 5

Chargaff

Answer 5

Hydrolysed DNA from different organisms and analysed proportion of bases.
[A] + [G] = [C] + [T]
[G] = [C], [A] = [T]

Question 6

Franklin and Wilkins

Answer 6

Exposed isolated fibres of DNA, exposed to X-ray beam and captured results of X-ray scattering on a plate.
Shows double strand wound around each other in a helical shape, spacing shows 3.4 Angstrom rise per bp, 20 A diameter.
Layer lines show not perfect helix.

Question 7

Watson and Crick

Answer 7

Used findings of Chargaff, Franklin and Wilkins as well as model building and proposed complementary base pairs (A to T w/ 2 H bonds, C to G w/ 3 H bonds), with similar widths.

Question 8

B-DNA (Predominant configuration)

Answer 8

• Complementary, anti-parallel strands.
• Right handed double helix (clockwise)
• Internal, flat hydrophobic bases with stacking interactions -> VdW forces contribute to stability
• Uneven spacing resulting in minor and major groove governing interaction with other molecules
• 2nm diameter, 10.5 bp per turn, 0.34 nm apart, 3.57 nm per turn

Question 9

Major vs minor groove

Answer 9

• Very easy to read chemical information in major groove differing by base; easily recognised by seq. spec. binding proteins (H bonds acceptors (A) and donors (D), methyl grps (M))
• In minor groove, T-A vs A-T, and G-C vs C-G present same grps so can’t be distinguished
• Non-seq. specific DNA binding proteins typically found in minor groove

Question 10

A and Z-DNA

Answer 10

• A:
  Low humidity. 2.6 nm diameter, right handed, 11bp/turn, EVEN sized grooves. Induced by DNA binding proteins.
• Z:
  1.8 nm diameter, left handed, 12bp/turn. Induced by methylation of C, torsional stress and high salt conc.

Question 11

Non B-DNA structures from repetitive seq.s

Answer 11

• Cruciform (Inverted repeats)
• Slipped/hairpin structure (direct repeat)
• Quadruplex (Oligo (G)n tracts)
  -> Hoogsteen base pairing

Question 12

Supercoiling

Answer 12

• Negative supercoiling supports easy unwinding
• Compacts
• Found in both linear and circular DNA