1. DNA Replication
August 30, 2017

2. Cell Cycle
DNA synthesis take place in S

3. Meselson-Stahl experiment
1958,
Matthew Meselson and Frank Stahl

4. Meselson-Stahl experiment

5. Meselson-Stahl experiment

6. Replication Fork Also predicted by Watson and Crick
Location the DNA double helix is unwound and produces two single strands that serve as templates for copying
John Cairns tested this prediction using thymidine with 3H (tritiated) to label newly synthesized daughter molecule

7. Replication Fork in E. coli
Autoradiograph is an image on an x-ray film or nuclear emulsion produced by the pattern of decay emissions (e.g., beta particles or gamma rays) from a distribution of a radioactive substance.
Autoradiograph is an image on an x-ray film or nuclear emulsion produced by the pattern of decay emissions from a distribution of a radioactive substance.

8. Discovery of DNA polymerase
Arthur Kornberg is credited with the discovery of DNA polymerase– using E. coli as a model system
The two reports describing the preparation and characterization of the four dNTPs, the partial purification of the deoxynucleotide kinases required for their synthesis, the partial purification of the newly named “DNA polymerase,” and the general properties of the polymerase reaction were declined by the Journal of Biological Chemistry when submitted in the Fall of 1957.
Among the critical comments were: “It is very doubtful that the authors are entitled to speak of the enzymatic synthesis of DNA”; “Polymerase is a poor name”; “Perhaps as important as the elimination of certain banalities...” etc.

9. DNA polymerases (pol)
First DNA polymerase isolated and activity was demonstrated in vitro in 1959 by Arthur Kornberg
To show activity: Add DNA
polymerase, deoxyribonucleotides (dATP, dGTP, dCTP and cTTP), a single strand of DNA template
There are 5 DNA pol in E. coli

10. DNA pol I Catalyze genes (DNA SYNTHESIS) in the 5’ to 3’ direction
Breaking the “high energy” phosphate bond provides energy to link the proximal phosphate to the growing chain
Synthesizes at ~10-20nt/second
Dissociates with DNA after incorporating 20 to 50 nt
This pol is NOT responsible for catalyzing DNA synthesis at the replication fork but is used in repair functions
3,000,000,000 *2 = 6x10^9 /400 = 1.5 * 10^7= 6250 min = 104 hrs =4.34 days

11. DNA pol III Catalyzes DNA synthesis at the replication fork
Replication fork is the zone where DNA is unwinding
DNA is always synthesized in the 5’ to 3’ direction
MUST have free 3’ hydroxyl for DNA synthesis to start

12. DNA Synthesis by Polymerases
What drives DNA synthesis?
The 3’ OH group mediates a nucleophilic attack of the alpha-phosphate, releasing a pyrophosphate group. DNA polymerase
DNA polymerase
Figure 9-2

13. DNA Synthesis by Polymerases
What drives DNA synthesis?
2. This reaction in coupled with pyrophosphate hydrolysis.
“High-energy” phosphate bonds: Hydrolysis releases energy and helps drives the reaction forward.
- Pyrophosphatases have been identified in bacteria and in eukaryotess (including cows) but not yet humans!
Figure 9-2
Pyrophosphatase

14. Origin of Replication
DNA helicase
DNA helicase binds to single stranded (ss) DNA and catalyzes the separation of the two strands in the double helix
ATP dependent

15. DNA Replication: Helicase
How can helicases unwind DNA in a directed fashion?
DNA helicase binds to ssDNA and catalyzes the separation of the two strands in the double helix
Moves directionally along ssDNA using ATP. It’s a motor!
Thought to “pull” the ssDNA through a central pore
Movement in the 5’-3’ direction or 3’-5’ direction
Acts at the replication fork
ssDNA

16. Eukaryotic DNA Replication

17. Topological Problem

18. DNA gyrase removes extra twists

19. Topoisomerases
50 revolutions
per second

20. Topoisomerase I Mechanism

21. Topoisomerase II Mechanism
-Double strand break
-ATP required

22. Topoisomerase II Inhibitors
They enhance the rate at which cleavage occurs and/or
reduces the rate at which the breaks are resealed