Mechanism of DNA Replication in Eukaryotes

Mechanism of DNA Replication in Eukaryotes

1. 1
DNA Replication
Matthew Meselson & Franklin Stahl, 1958
investigated the process of DNA replication
considered 3 possible mechanisms:
–conservative model
–semiconservative model
–dispersive model

2. (a) Hypothesis 1:
Semi-conservative
replication
(b) Hypothesis 2:
Conservative replication
Intermediate molecule
(c) Hypothesis 3:
Dispersive replication

3. 3

4. 4
DNA Replication
Bacterial cells were grown in a heavy
isotope of nitrogen,
15
N
all the DNA incorporated
15
N
cells were switched to media containing
lighter
14
N
DNA was extracted from the cells at various
time intervals

5. 5
DNA Replication
The DNA from different time points was
analyzed for ratio of
15
N to
14
N it contained
After 1 round of DNA replication, the DNA
consisted of a
14
N-
15
N hybrid molecule
After 2 rounds of replication, the DNA
contained 2 types of molecules:
–half the DNA was
14
N-
15
N hybrid
–half the DNA was composed of
14
N

6. 6

7. 7
DNA Replication
Meselson and Stahl concluded that the
mechanism of DNA replication is the
semiconservative model.
Each strand of DNA acts as a template for
the synthesis of a new strand.

8. 8
DNA Replication
DNA replication includes:
–initiation – replication begins at an
origin of replication
–elongation – new strands of DNA are
synthesized by DNA polymerase
–termination – replication is terminated
differently in prokaryotes and
eukaryotes

9. 9
Prokaryotic DNA Replication
The chromosome of a prokaryote is a
circular molecule of DNA.
Replication begins at one origin of
replication and proceeds in both
directions around the chromosome.

10. 10

11. 11
Prokaryotic DNA Replication
The double helix is unwound by the enzyme
helicase
DNA polymerase III (pol III) is the main
polymerase responsible for the majority of
DNA synthesis
DNA polymerase III adds nucleotides to the
3’ end of the daughter strand of DNA

12. 12

13. 13
Prokaryotic DNA Replication
DNA replication is semidiscontinuous.
–pol III can only add nucleotides to the 3’
end of the newly synthesized strand
–DNA strands are antiparallel to each other
leading strand is synthesized continuously (in
the same direction as the replication fork)
lagging strand is synthesized discontinuously
creating Okazaki fragments

14. 14

15. 15
Prokaryotic DNA Replication
The enzymes for DNA replication are
contained within the replisome.
The replisome consists of
–the primosome - composed of primase
and helicase
–2 DNA polymerase III molecules
The replication fork moves in 1 direction,
synthesizing both strands simultaneously.

16. 16

17. 17
Eukaryotic DNA Replication
The larger size and complex packaging of
eukaryotic chromosomes means they
must be replicated from multiple origins of
replication.
The enzymes of eukaryotic DNA replication
are more complex than those of
prokaryotic cells.

18. 18
Eukaryotic DNA Replication
Synthesizing the ends of the chromosomes
is difficult because of the lack of a primer.
With each round of DNA replication, the
linear eukaryotic chromosome becomes
shorter.

19. 19

20. 20
Eukaryotic DNA Replication
telomeres – repeated DNA sequence on
the ends of eukaryotic chromosomes
–produced by telomerase
telomerase contains an RNA region that is
used as a template so a DNA primer can
be produced

21. 21

22. 22
DNA Repair
- DNA-damaging agents
- repair mechanisms
- specific vs. nonspecific mechanisms

23. 23
DNA Repair
Mistakes during DNA replication can lead to
changes in the DNA sequence and DNA
damage.
DNA can also be damaged by chemical or
physical agents called mutagens.
Repair mechanisms may be used to correct
these problems.

24. 24
DNA Repair
DNA repair mechanisms can be:
–specific – targeting a particular type of
DNA damage
•photorepair of thymine dimers
–non-specific – able to repair many
different kinds of DNA damage
•excision repair to correct damaged
or mismatched nitrogenous bases

25. 25

26. 26

27. The 10 subunits of E. coli DNA polymerase III
SubunitFunction







’


5’ to 3’ polymerizing activity
3’ to 5’ exonuclease activity
 and  assembly (scaffold)
Assembly of holoenzyme on DNA
Sliding clamp = processivity factor
Clamp-loading complex
Clamp-loading complex
Clamp-loading complex
Clamp-loading complex
Clamp-loading complex
Core
enzyme
H
o
l
o
e
n
z
y
m
e
DNA Polymerase III Holoenzyme (Replicase)