1. REPLICATION
© 2016 Paul Billiet ODWS

2. A complex reaction Uncoiling of parent molecule
Unzipping the two sister strands to reveal the sequence of bases
Reading the sequence of bases
Choosing the complementary nucleotide building blocks
Lining up the nucleotides and bonding them together
Checking for errors
Recoiling the two DNA molecules.
All controlled by enzymes in particular DNA polymerase
© 2016 Paul Billiet ODWS

3. DNA polymerase III
© 2016 Paul Billiet ODWS

4. A very rapid reaction
The average length the DNA molecule in a bacteriophage (a large virus) is 34µm long
base pairs
turns (10 base pairs per revolution)
Replication time 2 minutes
Replication speed 83 revolutions per second.
© 2016 Paul Billiet ODWS

5. Multiple replication forks
Eukaryotes have much more DNA
Their DNA is divided up into many molecules (chromosomes)
Replication in eukaryotes begins at many points along each chromosome
This reduces the time taken.
© 2016 Paul Billiet ODWS

6. Where and when does replication occur?
In the nucleus of eukaryotes
During interphase
During S-phase
© 2016 Paul Billiet ODWS

7. Cytokinesis division of the cytoplasm
The cell cycle
Cytokinesis division of the cytoplasm
Some cells may stay in this stage for over a year G0 M
First growth phase.
Varies in length
Mitosis G1 G2
Interphase
Second growth period
Copying of chromosomes
= replication S
G1 + S + G2 = INTERPHASE
© 2016 Paul Billiet ODWS

8. Meselson & Stahl’s experiment
Samples taken at timed intervals
And DNA extracted
ultracentrifuge
Bacteria fed on N-15 labelled food for several generations
Bacteria switched to N-14 labelled food
DNA settles a level because of its density
© 2016 Paul Billiet ODWS

9. Meselson and Stahl’s results
DNA
Light Medium Heavy
Controls
GENERATIONS
© 2016 Paul Billiet ODWS

10. Observations Initially all the DNA is “heavy” Only one band appears
After one generation there is one band but it is “medium”
After two generations there are two equal bands “Medium” and “Light”
After three generations there are two bands A strong light band and a weaker medium
This carries on, the light band getting stronger.
© 2016 Paul Billiet ODWS

11. Interpretation of the results
GENERATION 1 2 3
© 2016 Paul Billiet ODWS

12. Interpretation At each generation the DNA molecule splits
A new strand is fabricated alongside the old one
This is semi-conservative replication.
© 2016 Paul Billiet ODWS

13. E.coli caught in the act! Newly formed daughter strands Growing point
2 strands of parental DNA
Growing point
© 2016 Paul Billiet ODWS

14. Untwisting the helix & breaking the hydrogen bonds
A = T
T = A
C  G
G  C
T = T A G T
A = T
T = A
C  G
G  C C
Helicase +
DNA gyrase +
single strand binding proteins,
© 2016 Paul Billiet ODWS

15. Adding in the nucleotides
A = T
T = A
C  G
G  C T C A A G T
DNA
Polymerase III A T
Deoxynucleoside triphosphates
Complementary base pairing
© 2016 Paul Billiet ODWS

16. Two daughter strands A = T A = T T = A T = A C  G C  G G  C G  C
© 2016 Paul Billiet ODWS

17. Added complications
DNA polymerase III cannot start the process of replication
A small primer of RNA is needed first
This requires another enzyme DNA primase.
© 2016 Paul Billiet ODWS

18. A = T T = A C  G G  C DNA T Polymerase III G C A G T A A T
DNA primase
© 2016 Paul Billiet ODWS

19. Added complications
DNA polymerase III can only add nucleotides on one way (5’ to 3’)
BUT the DNA molecule is antiparallel
One strand can be replicated directly as it unzips (the leading strand)
The other strand needs to wait until a certain amount is unzipped (the lagging strand).
© 2016 Paul Billiet ODWS

20. 5’ 3’ Okazaki fragments 3’ 5’ 5’ 5’ 3’ 3’ 5’ Leading strand
Lagging strand
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21. Added complications
The lagging strand is replicated in fragments about 1000 base pairs long
OKAZAKI fragments
Each fragment starts with an RNA primer.
© 2016 Paul Billiet ODWS

22. Added complications
At the end the RNA primers are removed by another enzyme, DNA polymerase I
Replaces the primers with DNA nucleotides
The ends of the Okazaki fragments are stuck together using DNA ligase.
© 2016 Paul Billiet ODWS

23. Ligase
DNA polymerase I
© 2016 Paul Billiet ODWS