DNA replication

• DNA replication

  The process by which a cell makes an exact copy of its DNA
• DNA replication

  • Every cell cycle the entire genome must be replicated precisely
  • Replication and cell division are not independent
  • It passes on a copy of its genetic material to its next generation
• DNA replication


  The process by which a cell makes an exact copy of its DNA
• DNA replication


  • Every cell cycle the entire genome must be replicated precisely
  • Replication and cell division are not independent
  • It passes on a copy of its genetic material to its next generation

  
• James Watson and Francis Crick

  Proposed the model of the structure of DNA

  
• Watson and Crick published their model of the structure of DNA in the journal Nature


  April 25th 1953
• Watson and Crick's model of DNA structure

  1. Two polynucleotide chains wound around each other in a right-handed double helix

  2. Two chains have opposite polarity

  3. The sugar-phosphate backbone is on the outside, with bases in the centre

  4. Bases are bonded together with hydrogen bonds, A with T and G with C

  5. Bases are 0.34 nm apart

  6. There is a major groove and a minor groove

  
• The structure of DNA double helix was the key to how it replicates
• Parent and daughter DNA

  • The original DNA duplex is the parent
  • The newly formed duplexes are daughters
  • When it starts to separate/unzip by braking down hydrogen bonds, the new strands are the daughter molecules

  
• Models of DNA replication

  • Semiconservative
  • Conservative
  • Dispersive

  
• Semiconservative replication


  • Parental duplex unwinds each strand acts as a new template for new strand synthesis
  • The parental duplex separates and we get the 1st generations which is a mix of an old strand and a new strand
  • 2nd generation is 50% new and 50% old/new mixed duplex

  
• Conservative replication


  • Parental duplex replicates to give 1 new and 1 parental duplex in generation 1
  • The original parental strands come together and the new strands that have been synthesized come together
  • No mixing of old and new DNA strands together
  • 2nd generation has 3 new and 1 parental duplex

  
• Dispersive replication


  • Parental duplex replicates to give duplexes consisting of new parts and parental parts
  • No strand is entirely new or parental
  • Each strand is mixed up of new/old pieces – fragmented copy of DNA
  • Generations 1 and 2 have similar composition

  
• Meselson and Stahl set out to test the alternative hypotheses for DNA replication in 1958

  
• Labelling DNA with "heavy" nitrogen

  • DNA contains nitrogenous bases, the nucleotide bases, and they were rich in nitrogen
  • Nitrogen comes in different forms, radioactive forms (radioactive) and other forms (non-radioactive)
  • N15 is literally heavier than N14

  
• Centrifugation to separate N14 from N15

  1. Equilibrium centrifugation of DNA of different densities in a caesium chloride density gradient

  2. Put caesium chloride and put the sample of DNA into that tube, then run it in an ultracentrifuge, the DNA will separate based on its mass

  3. If the DNA contains the N15 it will go down further in the tube as it is heavier

  
• Generation 0


  • Meselson and Stahl grew bacteria in a medium containing only the heavy isotope of Nitrogen – N15
  • All the DNA should now be heavy
• Meselson and Stahl experiment

  1. E. coli were initially grown in medium containing only the heavy N15

  2. The cells were transferred to a medium containing only the light N14 form

  3. DNA replicated in this medium would contain a mixture of N14 and N15

  4. Some of the cells were grown for further rounds of replication in medium containing only N14

  5. DNA was isolated from all of the cultures at time points representing a series of bacterial generations and tested for the composition of N14 and N15

  
• Predictions of the 3 DNA replication models

  • Semiconservative replication predictions
  • Conservative replication predictions
  • Dispersive replication predictions

  
• The results fit the semiconservative model of DNA replication
• Replicons


  • Replication of DNA molecules within cells takes place in segments known as replicons
  • A replicon is a unit of DNA in which individual acts of replication occur
  • Each replicon fires once (and no more) per cell cycle
  • Each has an origin where replication initiates and a terminus where it stops
  • Any sequence not separated from an origin by a terminus is replicates as part of the replicon

  
• Replicons in bacteria

  • Bacteria have a single origin of replication for the entire genome
  • Initiation occurs once per cell cycle
• Replicons in eukaryotes


  • Eukaryotes have a large number of replicons
  • Multiple replicons may be advantageous because eukaryotes have much more DNA
  • If all replicons fired at once replication could finish within 1 hour
  • Actually, takes about 6-8 hours
  • Maybe 15% are active at one time
  • Eukaryotic origin sequences are poorly understood
• A general feature of all origins of replication (eukaryotes too) is a high frequency of A + T nucleotides