unit 3

• DNA Replication and Repair

  The process of semiconservative replication of DNA
• Due to the work of Watson, Crick, Franklin, and Wilkins, an accurate model of DNA was determined in the 1950s
• DNA structure


  • Double helix structure, with the "sides" consisting of alternating deoxyribose sugars and phosphates
  • The "rungs" consist of nucleotide base pairs (A, T, C, &G)
  • The strands run antiparallel to each other
  • The hydroxyl on the 3' carbon of deoxyribose is at one end of the strand
  • The phosphate on the 5' carbon is at the other end
  • The strands run in opposite directions
• Meselson and Stahl verified that DNA replication was semiconservative


  1958
• Meselson and Stahl's Experiment

  1. Used "heavy" isotopes of nitrogen (15N) to label E. coli bacteria (lots of N in DNA!)

  2. Then transferred colonies to a growth medium of normal N, allowed to replicate for one or two rounds :. New DNA would contain "light" N and density could be measured
• Eukaryotic DNA replication is similar to prokaryotic, but more complex due to its linear configuration and sheer volume
• DNA Replication


  1. Strand separation

  2. Building complementary strands

  3. Dealing with errors
• Strand Separation


  1. DNA helicase binds to specific nucleotide sequences (replication origins)

  2. Unwinds DNA by breaking apart H-bonds between base pairs

  3. Replication fork: Y-shaped region of separation

  4. Tension on DNA behind fork (topoisomerase)

  5. Separated strands tend to anneal (SSBPs)

  6. Helicase will separate strands in both directions, forming a replication bubble

  7. There can be many replication bubbles at any given time on a strand of DNA; they will extend until they meet and merge

  8. DNA is replicated at a rate of ~50bp per second at each fork

  9. It takes about an hour to replicate the entire genome
• Building Complementary Strands


  1. DNA polymerases are enzymes that add nucleotides to build new DNA strands

  2. Nucleotides are added to the 3' end of the existing "template" strand, which is read in the 3' to 5' direction

  3. New strand: 5` 3`

  4. DNA polymerases need energy, which comes from the hydrolysis of 2 Pi from a nucleoside triphosphate as it is added to the strand

  5. Nucleoside = Sugar + Base, Nucleotide = Sugar + Base + Phosphate

  6. DNA polymerase III can only add to the 3' end of a strand, so RNA primase builds a short (10 – 60 bp) complementary RNA sequence called an RNA primer

  7. DNA polymerase III begins adding to the primer in the 5' to 3' direction

  8. One strand will be able to be synthesized continuously: leading strand

  9. The other side must be made in smaller fragments, using multiple RNA primers: lagging strand

  10. These DNA fragments on the lagging strand are called Okazaki fragments, 100-200 bp long in eukaryotes, 1000-2000 bp long in prokaryotes

  11. As each fragment extends, it will run into the RNA primer of the previous Okazaki fragment

  12. DNA polymerase I removes the RNA nucleotides and replaces them with those of DNA

  13. DNA ligase catalyzes the formation of a phosphodiester bond between the nucleotides of the two fragments
• Error Correction


  1. DNA polymerases also proofreads and corrects the newly synthesized strands

  2. For example, if there is a base pair mismatch (e.g. A and C), DNA polymerase III can't continue

  3. It will back up, replace the nucleotide, and continue

  4. Sometimes, errors will be missed (1 in every million bp) which distort the shape of DNA

  5. DNA polymerase II has a repair mechanism that can determine which is the original correct template strand, and remove the incorrect bases so they can be replaced
• Emperor Tamarin