DNA Replication

• DNA replication


  1. During the S phase of the cell cycle, DNA replicates

  2. A short section of the DNA uncoils

  3. New nucleotides bond to the open sections of the DNA

  4. The “new” and “old” sections of DNA separate and now there are 2 DNA strands (chromatids)

  5. Semiconservative model proposed by Watson and Crick, one of the two strands of every newly synthesized DNA molecule comes from the parent molecule

  6. Three steps: initiation, elongation, and termination
• DNA replication


  • Performed by multi-enzyme complex – DNA polymerases, Helicase, Primase, SSBs, DNA ligase, Clamps (Topoisomerases)
• DNA Replication - General aspects

  • Each original strand functions as a template for DNA synthesis
  • DNA is synthesized in 5’ to 3’ direction
  • DNA is synthesized by DNA polymerase
  • Replication fork is asymmetrical
  • Length of Okazaki fragments in prokaryotes are 1000-2000 nt, in eukaryotes 100-200 nt
• Proofreading mechanisms by DNA polymerase
• Error rate of 1 in 1 billion nucleotides
• DNA polymerase


  • Self-correcting enzyme with Polymerase activity, 3’ to 5’ exonuclease activity, 5’ to 3’ exonuclease activity
• DNA polymerase requirements


  • A free 3’-OH group supplied by RNA Primer for start of polymerisation
  • Mg2+ ions for activity in active site
  • A template to copy
• RNA primer synthesis


  1. Replication begins with a special RNA polymerase (primase) that synthesizes an RNA primer

  2. The RNA primer will be removed by the next molecule of DNA polymerase

  3. Ligase will finally join the newly synthesized DNA fragments
• DNA helicase


  • Enzyme involved in opening the DNA helix into its single strands for DNA replication
• DNA Topoisomerases


  • Prevent DNA tangling during replication
  • Relieve torsional stress caused by rotation of DNA ahead of the fork
  • Topoisomerase I
  • Topoisomerase II (DNA gyrase)
• DNA replication takes place at replication fork
• DNA polymerase catalyses nucleotide polymerisation in a 5’-3’ direction
• On the lagging strand, the short DNA fragments must be made – Okazaki fragments
• Lagging strand DNA primers are primed by short RNA primer
• Molecules needed for replication

  • DNA polymerase and DNA primase
  • DNA helicase and single-strand DNA binding proteins
  • DNA ligase
  • DNA topoisomerase
• DNA replication


  1. How replication machinery is assembled

  2. How cell starts the replication and how it is regulated

  3. Replication machinery in bacteria and eukaryotes

  4. "End of replication" problem in eukaryotes - telomerase
• Replication origins


  • DNA synthesis starts at replication origins
  • Bacteria has a single origin of replication
  • Eukaryotes have multiple origins of replication. In human cells, replication begins at 30,000-50,000 origins
• Replication initiation (Bacteria)


  1. Initiator protein = dnaA

  2. Helicase = dnaB

  3. Primase = dnaG
• E. Coli contains the 4.6X106 bp and it is a single circular DNA molecule
• DNA replication takes place during the cell cycle
• Phases of the cell cycle

  • Mitosis
  • G1-phase
  • S-phase
  • G2-phase
• Interphase is the period in between each mitotic cell division
• Average mammalian cell cycle in vitro is 24-26h but liver cells can take a year
• 1. phase lasts 6-8h
• 1. phase lasts 30-45min
• Average yeast cell cycle is 90-120 min
• G1 phase can last from 10 hours to minutes
• Length of the cell cycle has been studied by labelling nucleotides and measuring the rate of incorporation. Actually measured by FLOW CYTOMETRY
• Frequency of cell division varies by cell type
• Frequency of cell division by cell type

  • Embryo: cell cycle < 20 minutes
  • Skin cells: divide frequently throughout life, 12-24 hours cycle
  • Liver cells: retain ability to divide, but keep it in reserve, divide once every year or two
  • Mature nerve cells & muscle cells: do not divide at all after maturity, permanently in G0
• Synthesis of RNA and protein occurs continuously but DNA synthesis only occurs in the discrete period of S phase
• Phases of Mitosis


  • Prophase
  • Metaphase
  • Anaphase
  • Telophase
• ATP is synthesized during Mitosis
• Damaged parts are repaired during Mitosis
• Wastes are excreted during Mitosis
• Proteins are made during Mitosis
• Organelles are formed during Mitosis
• Chromosomes are copied during Mitosis
• Specialized tasks are performed during Mitosis
• Centrosomes duplicate during Mitosis