DNA Replication and Repair

• What is DNA replication and repair?

  The process by which DNA is copied and errors are corrected
• DNA Replication

  • DNA must be copied accurately and in a coordinated fashion when a cell divides
  • The antiparallel strands of DNA act as templates for each other
  • Specific base pairing allows for accurate copying
  • The tightly wound helical structure needs to be unwound for replication

  
• The stages of DNA replication

  1. Initiation

  2. Elongation

  3. Termination

  
• Initiation in Bacteria

  • DNA replication starts from one point of origin called the origin of replication
  • Specific proteins (initiation complex) recognise DNA sequences at the origin and begin to open out the helix and unwind it
  • This forms a replication bubble, which allows access to proteins that synthesise the new strands

  
• Eukaryotic DNA Replication


  • Eukaryotes have multiple origins of replication
  • Replication bubbles merge and coalesce, and replication occurs at many places in the genome
• Molecular Mechanisms of DNA Replication

  1. Incorporation of new nucleotides

  2. Elongation

  3. Discontinuous Replication

  
• Incorporation of New Nucleotides

  Nucleotide triphosphate is attacked by the OH group of the ribose, forming a phosphodiester bond and releasing pyrophosphate
• Elongation


  • DNA elongates in a 5' to 3' direction
  • DNA polymerase is the key enzyme responsible for elongation

  
• Discontinuous Replication


  One strand (leading strand) can be copied continuously, the other strand (lagging strand) needs to be replicated in small sections

  
• DNA Polymerase


  • There are several types, each with specific roles
  • Cannot de novo synthesise DNA strands, must have a template and can only extend on the 3' end
  • Require a primer, nucleotide triphosphates and magnesium ions

  
• DNA Primase


  Synthesizes short RNA sequences called primers, which serve as a starting point for DNA synthesis

  
• Lagging Strand


  • DNA synthesis has to be continuously re-initiated, resulting in Okazaki fragments
  • Okazaki fragments are short DNA sequences formed discontinuously, initiated by new RNA primers
  • Ligase enzyme joins the Okazaki fragments together

  
• Key Factors for DNA Replication

  • Helicase unwinds the DNA at the origin of replication
  • Primase adds RNA primers
  • DNA polymerase synthesizes the leading strand continuously and the lagging strand discontinuously
  • RNase H removes the RNA primers and DNA ligase fills the gaps

  
• Other Enzymes and Proteins Required

  • DNA helicase
  • Single stranded DNA binding protein
  • Topoisomerase

  
• DNA Polymerase Fidelity

  • The ability of DNA polymerase to accurately replicate a template
  • Polymerases make errors approximately once every 10^4-10^5 nucleotides, but the actual error rate is closer to 1 in 10^9 or 1 in 10^10 due to proofreading and correction mechanisms

  
• Base Pairing Rules

  • Correct base pairing is more energetically favourable and preferred in the geometry of the polymerase active site
  • The polymerase has a 3' to 5' exonuclease activity that checks and corrects base pairing errors
• Sources of DNA Damage

  • Endogenous (replicative errors, oxidative damage, alkylation)
  • Exogenous (UV, pollution, carcinogens, radiation, chemotherapy)
• Types of DNA Damage

  • Bases can become oxidised, damaged, alkylated, deaminated or lost
  • Bases can become dimerised
  • DNA backbone can break (single or double strand breaks)
  • Strands can become crosslinked

  
• DNA Repair Mechanisms

  • Direct reversals
  • Nucleotide excision repair
  • Base excision repair
  • Mismatch repair
  • Recombinational repair
  • Non-homologous end joining

  
• Direct Reversals

  • UV-induced thymine dimers are directly repaired by a lyase enzyme
  • Methylated bases are repaired by enzymes that remove the methyl groups
• Nucleotide Excision Repair

  A short single-stranded DNA segment containing the lesion is removed, and the undamaged strand is used as a template to synthesize a complementary sequence

  
• Base Excision Repair

  Only the affected base is removed, leaving an apurinic/apyrimidinic site, which is then filled in by DNA polymerase and ligase
• Mismatch Repair

  Mechanism similar to nucleotide excision repair, but specifically targets mismatched base pairs between the two DNA strands
• Recombination Repair


  A complex process that uses sequences from a homologous piece of DNA to repair the damaged DNA

  
• Non-homologous End Joining

  • Binding together of two broken DNA ends, which may or may not be from the same DNA strands
  • Very prone to error