Ch2

• DNA replication


  The process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules
• DNA replication is one of the most basic processes that occurs within a cell
• DNA replication occurs during the interphase of the cell cycle, especially in the S phase
• Watson and Crick proposition about DNA replication

  1. Separation of the 2 strands

  2. Creation of two daughter strands by pairing of template nucleotides with new nucleotides using the a:t and g:c pairing rule

  3. Semi-conservative replication
• Modes of DNA replication

  • Semiconservative
  • Conservative
  • Dispersive
• Meselson-Stahl experiment


  1. Growing E.coli in medium containing heavy 15N isotope

  2. Switching to medium with light 14N isotope

  3. Extracting and purifying DNA

  4. Measuring DNA density using density gradient centrifugation
• Meselson and Stahl's experiment provided experimental proof of semi-conservative replication
• Mechanism of DNA replication

  1. Initiation

  2. Elongation

  3. Termination
• DNA replication


  Requires a template (parental DNA molecule), enzymes, and nucleotide triphosphates
• How does DNA replicate?

  1. Unwind (topoisomerase)

  2. Unzip (DNA helicase)

  3. Hold open (single-strand binding proteins)

  4. Base pairing (DNA polymerase)

  5. Joining nucleotides (DNA ligase)
• Replication results in two identical DNA molecules, with one new strand and one old strand
• Leading strand


  Nucleotides are added in the same direction as the replication fork
• Lagging strand


  Nucleotides are added in the opposite direction as the replication fork, creating Okazaki fragments
• Polymerase only works in 5' to 3' direction on both parent strands
• Replication in prokaryotes vs eukaryotes

  • Prokaryotes: Replication rate of 1500 nucleotides per second, takes 40 minutes to complete E.Coli genome
  • Eukaryotes: Replication rate of 10-100 nucleotides per second
• Cell cycle in prokaryotes


  Binary fission
• Stages of DNA replication

  1. Initiation

  2. Elongation

  3. Termination
• Initiation


  Replication begins at specific DNA sequences called origin of replications (oriC)
• Initiation process


  1. DnaA protein recognizes DnaA box sequences in oriC

  2. DnaA proteins melt the two strands and open the helix

  3. DnaB (Helicase)-DnaC complex forms prepriming complex

  4. DNA gyrase and SSB bind to prepriming complex to form priming complex

  5. Helicases unwind DNA further

  6. Primase synthesizes primers

  7. DNA polymerase III forms replisome
• Primase


  Essential enzyme for primer formation, forms primosome with additional proteins
• DNA replisome


  Multiprotein machine that performs parental strand separation and RNA-primed DNA synthesis on both strands
• Elongation on leading strand

  DNA polymerase III synthesizes DNA by adding 5'-P of deoxynucleotide to 3'-OH group, chain grows in 5'->3' direction
• Elongation on lagging strand

  1. Synthesis of Okazaki fragments

  2. Excision of RNA primer by DNA polymerase I

  3. Filling the gap by DNA polymerase I

  4. Joining of Okazaki fragments by DNA ligase
• E. coli DNA polymerases

  • DNA polymerase I (pol I)
  • DNA polymerase II (pol II)
  • DNA polymerase III (pol III)
• Exonuclease


  Enzyme that removes successive nucleotides from the end of a polynucleotide molecule
• Endonuclease


  Enzyme that cuts nucleic acid internally
• Termination


  1. Occurs at specific site opposite oriC

  2. Intertwined daughter molecules are unlinked by DNA gyrase
• Cell cycle in eukaryotes

  1. Interphase (G1, S, G2)

  2. Mitosis
• Eukaryotic origins of replication


  Not sequence specific, depend on chromatin structure
• Eukaryotic replication fork


  1. Helicases load onto possible origins before S phase

  2. Subset of origins activated during S phase

  3. Priming by DNA polymerase α and primase

  4. Leading strand synthesis by DNA polymerase ε

  5. Lagging strand synthesis by DNA polymerase δ
• Telomeres


  Specialized structures found on the ends of eukaryotic chromosomes, composed of specific sequences
• Telomeres shorten with each round of DNA replication
• Telomerase


  Enzyme that synthesizes the telomere repeat sequences, uses an internal RNA template
• Telomerase activity is developmentally regulated, high in early development/childhood, low in most adult somatic cells
• Telomerase contributes to cancer, as cancer cells divide rapidly and generally show activation of telomerase to maintain telomere length