1. Replication of DNA
Notes & Animations

2. Cell Cycle G1: Growth S: Synthesis of DNA (Replication)
G2: Growth, preparation of organelle for mitosis
M: Mitosis

3. How does DNA Replicate? Meselson and Stahl’s experiment:
Hypothesis 1: “conservative” replication
Hypothesis 2: “semi-conservative” replication
Hypothesis 3: “dispersive” replication

4. “Conservative” Replication

5. “Semi-Conservative” Replication

6. “Dispersive” Replication

7. Method
E. coli grown in 15N (heavy Nitrogen) medium. Allowed to reproduce for 17 generations. (Makes all DNA heavy.)
Then, transferred to 14N media & allowed to reproduce again (New DNA will be light)
Centrifuged (spun) so that “heavy” DNA sinks to bottom, “light” DNA stays on top.

8. Predictions “Conservative” Replication:
After 1 replication, 2 distinct bands
original “heavy” DNA at bottom, new “light” DNA on top
After 2 replications, same 2 bands, but more DNA in “light” band

9. Conservative Replication
Heavy DNA
Light DNA

10. Predictions “Semi-Conservative” Replication
After 1 generation, 1 band in middle.
After 2 generations, 1 light band, 1 middle band

11. Semi-Conservative Replication
Heavy DNA
Light DNA

12. Predictions “Dispersive” replication:
After 1 replication, band would be in the middle.
After 2 or more replications, band would be in the middle, but getting higher each time.
Always hybrid.

13. Dispersive Replication Prediction
Heavy DNA
Light DNA

14. Observations

15. Now we know… Replication consists of 3 steps: Initiation:
DNA helicase unwinds double helix by breaking H-bonds
Proteins hold DNA apart (if not, DNA would re-anneal – stick back together)
Replication begins in 2 directions from many origins in eukaryotes – speeds up process

16. Replication Bubbles

17. Replication: Step 2 Elongation: Requires 4 more enzymes (eukaryotes)
Primase
Makes RNA primer
DNA polymerase III
Can’t start from nothing – attaches to primer
Moves in a 5’ to 3’ direction continuously (adds nucleotides to 3’C)

18. Uh oh, what about 3’  5’?
Made in short fragments called Okazaki fragments
Consist of RNA primer, then DNA polymerase adds a short sequence, then start over
Called the “lagging” strand
5’  3’ called the “leading” strand

19. Elongation, continued…
DNA polymerase I
Replaces RNA primers with DNA nucleotides and proofreads the new strand
DNA ligase
Joins Okazaki fragments together

20. Proofreading
When mistake is found, it is cut out and replaced with correct base (Pol I)
Errors missed at a rate of 1/billion bases!

21. Step 3 of Replication Termination Helix re-anneals automatically.
Note: strands can’t be fully finished because DNA polymerase I cannot replace the primers at the ends of the DNA.
Ends of chromosomes are stretches of repetitive “junk” DNA called Telomeres
Telomeres extended by telomerase (expressed during meiosis, and by cancerous cells)
Chromosomes lose about 100 bases from this telomeric region every replication