DNA Replication

• DNA (or RNA) is ALWAYS synthesised in the 5' → 3' direction


  The parental template strands are said to be/run in the 3' → 5' direction

  
• Eukaryotic DNA Replication

  • Bidirectional
  • Multiple origins of replication

  
• Leading strand


  Continuously synthesised in its 5' → 3' direction

  
• Lagging strand


  Discontinuously synthesised in its 5' → 3' direction as Okazaki fragments
• DNA polymerase III (Pol III)


  Enzyme that synthesises a new DNA strand by adding nucleotides complementary to the parental template strands

  
• Primase


  Enzyme (a type of RNA polymerase) that makes an RNA primer = starting point for DNA polymerisation

  
• DNA ligase


  Joins the newly synthesized Okazaki fragments together (creates phosphodiester bonds)
• DNA pol III proofreading mechanism

  Checks the newly inserted nucleotide bases against the template

  
• Repair of DNA errors DURING DNA Replication


  3'to 5'exonuclease activity of DNA pol III removes incorrect bases

  
• Repair of DNA errors AFTER DNA Replication


  1. A variety of things can cause DNA damage or errors, e.g. incorrectly inserted bases, radiation damage, chemical modifications of bases

  2. These types of incorrect bases are removed by an ENDOnuclease

  
• Repair of DNA errors AFTER DNA Replication

  1. Damaged/incorrect DNA is removed by an endonuclease

  2. A DNA polymerase makes new DNA

  3. DNA ligase joins new DNA to existing DNA

  
• DNA error and damage repair systems

  • Correct incorrect or damaged nucleotide bases
  • Remove damage, including some flanking regions

  
• If not corrected, the DNA error becomes part of the DNA template and results in a permanent DNA change/mutation
• All future DNA molecules arising from this 'mutated' DNA will carry the incorrect DNA base

  
• Polymerase Chain Reaction (PCR)

  Molecular photocopying
• PCR

  • In vitro method of making multiple DNA copies
  • Only 'targeted' DNA region will be copied
  • Rapid exponential increase of DNA molecules
  • Method utilises cycles of heating and cooling

  
• Applications of PCR


  • Medical
  • Forensic
  • Infectious disease detection and identification
  • Molecular biology research
• How PCR works

  1. 94 - 98°C

  2. 72°C

  3. 45 - 70°C
• DNA template


  DNA molecule to which complementary nucleotides can be matched to make identical copies via DNA synthesis

  
• Primers


  • Provide a free 3' OH group to initiate DNA synthesis
  • Define the region of the DNA molecule to be replicated

  
• Proteins involved in DNA replication

  • Primase
  • DNA polymerase III
  • Helicase
  • Topoisomerase
  • Single-stranded DNA binding proteins
  • DNA polymerase I (two activities)
  • DNA ligase

  
• Lagging strand


  Needs to be synthesised as smaller fragments, called Okazaki fragments
• Human (complex eukaryote)

  Has multiple chromosomes
• Eukaryote


  Has multiple origins of replication
• Exonuclease


  Enzyme that removes nucleotides from the end of a DNA strand
• Endonuclease


  Enzyme that cuts the DNA strand at internal sites

  
• Compenents required for PCR:

  Template DNA, primers (forward and reverse), dNTPs (deoxyribonucleotide triphosphates – A T, G and C), and DNA polymerase.
• Primers determine which region of DNA is synthesised by PCR.
• Process of PCR:

  Starting from the 3’ end of the primer, DNA polymerase adds in complementary bases/nucleotides to extend/synthesise the new strand in the 5’ to 3’ direction.

  • involved denaturing, annealing and extension
• • Joining of ends of newly synthesised fragments together (lagging as well as leading strands, within and between replication bubbles) (DNA ligase)
• DNA replication:

  Progressive addition of new nucleotide bases by DNA Polymerase III.
• The unwinding of the double stranded DNA is done by Helicase to produce two parental template.
• Topoisomerase releases tension by unwinding DNA strand and putting them back together.
• SSBP- Single Stranded Binding Protein prevents unwound DNA strands to reform and protect it from degradation.
• DNA Polymerase 1 recognises DNA-RNA hybrid and removes primer. It also uses the 3' OH end of one nucleotide and extend it to another nucleotide.
• DNA Ligase joins okazaki fragments via phosphodiester bonds to form a continuous strand.