1 - DNA Structure and Supercoiling

• In 1928, Fred Griffith demonstrated that nucleic acids isolated from heat-killed virulent bacteria could transform non-virulent bacteria into a virulent form, calling them the "transforming principle"
• Griffith used Streptococcus pneumoniae bacteria in his 1928 experiments to discover the "transforming principle"
• In 1944, a follow-up experiment to Griffith's was performed by Oswald Avery, Colin MacLeod and Macyln McCarty who identified that the transforming principle was DNA
• Avery's 1944 experiment involved:

  • Fractionated material isolated from heat-killed bacteria demonstrated that nucleic acids were the "transforming principle"
  • Transforming activity was destroyed when nucleic acids were treated with deoxyribonuclease (digests DNA) but not ribonuclease (digests RNA)
  • This identified Griffith's "transforming principle" as DNA
• In 1952, DNA was confimed as the genetic material by Alfred Hershey and Martha Chase
• In 1952, Hershey and Chase's experiment involved:

  • Labelling T2 bacteriophages with either 35S or 32P isotopes
  • Only 32P was detected in infected bacteria and in phage progeny
  • 35S isolated in phage "ghosts" that failed to enter the bacteria
  • This confirmed DNA as the genetic material
• DNA is the genetic material in nearly all cells
• Viruses contain mostly RNA
• A polynucleotide strand is made of nucleotides joined together by phosphodiester bonds that link the 3' OH of one sugar + phosphate to the 5' OH on the phosphate group of the next sugar
• Polynucleotides are directional and are conventially written in the 5' --> 3' direction
• Nucleotide = pentose sugar + base + phosphate group
• Bases are planar rings that are typically uncharged under physiological conditions
• DNA contains deoxribose

  RNA contains ribose

  They differ on the group of the 2' carbon

  
• RNA is more reactive than DNA because of the hydroxyl (OH) group on the 2' carbon of ribose and has catalytic functions
• Pyrimidines: cytosine, thymine, uracil

  
• Purines: adenine, guanine

  
• In the pyrimidines (C, T, U) the base attaches to the 1' C of the sugar from N1 via a glycosidic bond
• In the purines (A, G) the base attaches to the 1' C of the sugar from N9 via a glycosidic bond
• Bases can exist as tautomers (a molecule where a proton has migrated to a different place)
• 0.0033% of the human genome (3 Gbp) are rare tautomers

  
• Genetic variation can arise from rare tautomeric forms of bases being replicated
• Nucleoside = base + sugar
• Phosphate groups are added to the C5' of the sugar to form a nucleotide
• The glycosidic bonds that attaches a base to a sugar is formed by a condensation reaction
• Examples of nucleotide monomers that are often donors:

  • ATP
  • CoA
  • S-adenosyl methionine
  • NAD
  • FAD
• Erwin Chargaff studied base composition in DNA by:

  • Purifying DNA from a range of organisms
  • Hydrolysed the DNA to free the bases
  • Analysed the proportions of the bases
  • Results: [A]+[G]=[C]+[T] (amount of purine bases = pyramidine bases)
• Watson and Crick incorporated the work of Chargaff, Franklin & Wilkins into their model
• Watson and Crick incorporated the work of Chargaff, Franklin & Wilkins into their model
• Rosalind Franklin and Maurice Wilkins used X-ray diffraction of DNA fibres to discover the double helix structure of DNA
• Franklin and Wilkins discovered:

  • The helical structure of DNA was characterised by 2 intertwined helices
  • The line spacings suggested the dimenstions of the Helix:
  • 34 Armstrongs (3.4 nm) and ~10 bp per turn
  • 3.4 Armstrongs (0.34 nm) rise per bp
  • 20 Armstrongs (2 nm) helix diameter
• Franklin and Wilkins' experiment involved shining X-ray beams onto DNA fibres at high humidity which scattered into the arrangement of atoms/molecules in the DNA sample which can be captured on a photographic plate
• The double helix structure of DNA was proposed by Watson and Crick in 1953
• Watson and Crick proposed that there would be 2 polynucleotide strands that were associated via weak hydrogen bonds to form double-stranded DNA
• Watson and Crick discovered that A & T paired with 2 H bonds, while C and G paired with 3 H bonds

  
• DNA that needs to be opened up for initiating replication or transcription is AT pair rich as there are fewer hydrogen bonds to break
• Watson and Crick's proposal of the structure of hydrated B-DNA:

  • Two complementary DNA strands are antiparallel and wind around each other in a right-handed double helix (clockwise)
  • Hydrophilic sugar phosphate backbone is on the outside of the molecule
  • Hydrophobic bases form a stack on the interior of the helix
  • Van der Waals forces between bases stabilise interactions (and DNA as a whole)
  • The strands are not wrapped around each other equally (there is a major groove and minor groove)
  • Contribution of base stacking to stability varies with sequence
• The structure of B-DNA was confirmed in 1980 by analysing its crystal structure
• 1980 discovery of B-DNA structure:

  • Helix diameter = ~2 nm
  • 10.5 bp in one complete turn of the helix
  • Base pairs are 0.34 nm apart
  • One full turn of the helix is 3.57 nm
  • The helix forms a major and minor groove
  • B-DNA is the predominant configuration in cells
• The major groove is rich in chemical information (such as hydrogen bond acceptors & donors and methyl groups)
• In the minor groove, AT and GC base pairs are both acceptor-donor-acceptor (present the same chemical groups) and can't be distinguished