Meselson & Stahl In 1950, Matthew Meselson and Franklin Stahl grew E.coli bacteria in a 15 N rich environment. They then transferred the E.coli into a 14 N rich environment. After three generations, Meselson and Stahl used centrifugation to isolate the DNA of each generation of E.coli and compare their densities

Meselson & Stahl Their results: The first generation (grown in 15 N) had heavy DNA The second generation ( 15 N E.coli grown in 14 N) had intermediate DNA The third generation (still in 14 N) had both intermediate and light DNA Conclusion: E.coli separate their DNA using semiconservative replication!

Semiconservative Replication Even though Meselson and Stahl thought it only applied to bacteria, other researchers found that it occurs in eukaryotes as well! But what is Semiconservative Replication? Individual strands of DNA stay intact and are used as templates for replication of new strands of complimentary DNA This means each new strand of DNA is half “old” and half “new”.

Replication & Repair Separation DNA helicase is an enzyme that unwinds the DNA by breaking the hydrogen bonds between base pairs To keep it apart, single-stranded binding proteins (SSBP) bind to the strands to prevent base pairs from re-annealing (pairing of complimentary strands of DNA through hydrogen bonding) DNA gyrase relieves tension from unwinding When an enzyme reaches a point of untwisted, single- stranded DNA, we call it a replication fork. John Cairns found that DNA replicated bidirectionally.

Evidence points to bidirectional replication Label at both replication forks

Replication & Repair Building Complimentary Strands An RNA primer anneals to the template strand and is synthesized by primase (enzyme). DNA polymerase III binds to the RNA primer and elongates the strands of DNA It synthesizes a new DNA strand in the 5’ to 3’ direction by adding complimentary nucleotides to the 3’ end of the growing strand. Arthur Kornberg found the first DNA polymerase enzyme and was the first to discover they only added nucleotides in the 5’ to 3’ direction.

Replication & Repair DNA is always synthesized in the 5’ to 3’ direction Since DNA strands are antiparallel, only one strand can be synthesized continuously The leading strand is synthesizes towards the replication fork, and therefore is continuous The lagging strand however, is being synthesized away from the replication fork  lagging strand  leading strand

Replication & Repair RNA primers are constantly being added to the lagging strand to allow the forming of pieces of synthesized DNA called Okazaki Fragments (after Reija Okazaki who discovered them) DNA polymerase I or Rnase H removes the RNA primers (from both the strands) and replaces them with the appropriate nucleotides. DNA ligase joins the Okazaki fragments together into one strand by creating a phosphodiester bond. As both strands are synthesized, they automatically twist back into a double helix.

Replication & Repair Quality Control: Repair! If mistakes in replication are found, DNA polymerase I and III can act as an exonuclease They will excise an incorrectly paired nucleotide Mistakes must be made immediately in order to prevent them from being copied in future replications Replication and Repair Video!

DNA Replication Tutorial nt/animations/dna_replication/index.html nt/animations/dna_replication/index.html This is awesome!!! Do this!!!