1. DNA Replication

2. DNA is the Genetic Material
Therefore it must
Replicate faithfully.
Have the coding capacity to generate proteins and other products for all cellular functions.
“A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.”
-Francis Crick

3. Replication
Watson and Crick develop the double helix model of DNA

4. The Dawn of Molecular Biology
April 25, 1953
Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." 3

5. Testing Models for DNA replication
Matthew Meselson and Franklin Stahl (1958)

6. Models for DNA replication
1) Semiconservative model:
Daughter DNA molecules contain one parental
strand and one newly-replicated strand
2) Conservative model:
Parent strands transfer information to an
intermediate (?), then the intermediate gets copied.
The parent helix is conserved, the daughter
helix is completely new
3) Dispersive model:
Parent helix is broken into fragments, dispersed, copied then assembled into two new helices.
New and old DNA are completely dispersed

7. MODELS OF DNA REPLICATION
(a) Hypothesis 1:
(b) Hypothesis 2:
(c) Hypothesis 3:
Semi-conservative replication
Conservative replication
Dispersive replication
Intermediate molecule

8. Meselson and Stahl Semi-conservative replication of DNA
Isotopes of nitrogen (non-radioactive) were used in this experiment

9. Bases paired Strands antiparallel

10. TEMPLATING/REPLICATION
REPLICATION OF INFORMATION

11. Key proposal of Watson and Crick: base pairs A : T and G : C
are specific. Base pairing regulates replication.

12. Replication is a Process
Double-stranded DNA unwinds.
The junction of the unwound
molecules is a replication fork.
A new strand is formed by pairing
complementary bases with the
old strand.
Two molecules are made.
Each has one new and one old
DNA strand.

13. Replication DNA molecule separates into 2 strands
Each strand serves as a template for a new strand
Complementary strand is made

14. Replication and Enzymes
Enzymes = proteins helping to catalyze (start and speed up) a reaction
Main enzyme in replication = DNA polymerase
DNA polymerase
Joins individual nucleotides to make DNA
“proofreads” new DNA strands to check for error

15. DNA replication
Nucleotides are successively added using deoxynucleoside triphosphosphates (dNTP’s)

16. Replication can be Uni- or Bidirectional
Origin 5’ 3’
UNIDIRECTIONAL REPLICATION
Origin 5’ 3’
BIDIRECTIONAL REPLICATION

17. T7 DNA replication 1 Replicating bubble in DNA from bacteriophage T7
Two replication forks heading towards opposite ends of the DNA

18. Features of DNA Replication
DNA replication is semiconservative
Each strand of both replication forks is being copied.
DNA replication is bidirectional
Bidirectional replication involves two replication forks, which move in opposite directions

20. Arthur Kornberg (1957)
Protein extracts from E. coli +
Template DNA
Is new DNA synthesized??
- dNTPs (substrates) all 4 at once
- Mg2+ (cofactor)
- ATP (energy source)
- free 3’OH end (primer)
In vitro assay for DNA synthesis
Used the assay to purify a DNA polymerizing enzyme
DNA polymerase I

21. -therefore DNA synthesis occurs only in the
Kornberg also used the in vitro assay to characterize
the DNA polymerizing activity
- dNTPs are ONLY added to the 3’ end of newly
replicating DNA 5’ 3’
Parental template strand
New progeny strand 5’ 3’ 3’ 5’ 3’ 3’ 5’ 3’ 3’
-therefore DNA synthesis occurs only in the
5’ to 3’ direction

22. THIS LEADS TO A CONCEPTUAL PROBLEM
Consider one replication fork: 5’ 3’ 5’ 3’
Primer
Continuous replication
Direction of
unwinding
Primer 5’ 3’
Discontinuous replication
Primer 5’ 3’

23. Evidence for the Semi-Discontinuous replication
model was provided by the Okazaki (1968)

24. Evidence for Semi-Discontinuous Replication (pulse-chase experiment)
Bacteria are
replicating
Bacterial
culture
Add 3H Thymidine
For a SHORT time
(i.e. seconds)
Flood with non-radioactive T
Allow replication
To continue
Harvest the bacteria
at different times
after the chase
smallest
largest
Isolate their DNA
Separate the strands
(using alkali conditions)
Run on a sizing gradient
Radioactivity will only
be in the DNA that was
made during the pulse

25. Results of pulse-chase experiment5’ 3’
Direction of
unwinding
Primer
smallest
largest *
Primer 5’ 3’ *

26. DNA replication is semi-discontinuous
Continuous synthesis
Discontinuous synthesis

27. Features of DNA Replication
DNA replication is semiconservative
Each strand of template DNA is being copied.
DNA replication is bidirectional
Bidirectional replication involves two replication forks, which move in opposite directions
DNA replication is semidiscontinuous
The leading strand copies continuously
The lagging strand copies in segments (Okazaki fragments) which must be joined

28. The Enzymology
of DNA Replication
In 1957, Arthur Kornberg demonstrated the existence of a DNA polymerase - DNA polymerase I
DNA Polymerase I has THREE different enzymatic activities in a single polypeptide:
a 5’ to 3’ DNA polymerizing activity
a 3’ to 5’ exonuclease activity
a 5’ to 3’ exonuclease activity 5

29. The 5’ to 3’ DNA polymerizing activity
Subsequent
hydrolysis of
PPi drives the
reaction forward
Nucleotides are added at the 3'-end of the strand

30. Why the exonuclease activities?
The 3'-5' exonuclease activity serves a proofreading function
It removes incorrectly matched bases, so that the polymerase can try again. 7

31. Proof reading activity
of the 3’ to 5’ exonuclease.
DNAPI stalls if the incorrect
ntd is added - it can’t add the
next ntd in the chain
Proof reading activity is slow
compared to polymerizing
activity, but the stalling of
DNAP I after insertion of an
incorrect base allows the
proofreading activity to
catch up with the polymerizing
activity and remove the
incorrect base.

32. DNA Replication is Accurate (In E. coli: 1 error/109 -1010 dNTPs added)
How?
1) Base-pairing specificity at the active site
- correct geometry in the active site occurs only with correctly paired bases BUT the wrong base still gets inserted 1/ dNTPs added
2) Proofreading activity by 3’-5’ exonuclease
- removes mispaired dNTPs from 3’ end of DNA
- increases the accuracy of replication fold
3) Mismatch repair system
- corrects mismatches AFTER DNA replication

33. Why the exonuclease activities?
The 5’-3' exonuclease activity is used to excise RNA primers in a recation called “nick translation”
We will discuss this in the next lecture. 7

34. Is DNA Polymerase I the principal replication enzyme??
In 1969 John Cairns and Paula deLucia isolated a mutant bacterial strain with only 1% DNAP I activity (polA)
- mutant was super sensitive to UV radiation
- but otherwise the mutant was fine i.e. it could divide, so obviously it can replicate its DNA
Conclusion:
DNAP I is NOT the principal replication enzyme in E. coli 9

35. Other clues….
- DNAP I is too slow (600 dNTPs added/minute – would take 100 hrs to replicate genome instead of 40 minutes)
- DNAP I is only moderately processive
(processivity refers to the number of dNTPs added to a growing DNA chain before the enzyme dissociates from the template)
Conclusion:
There must be additional DNA polymerases.
Biochemists purified them from the polA mutant 9

36. So if it’s not the chief replication enzyme then what does DNAP I do?
- functions in multiple processes that require only short lengths of DNA synthesis
- has a major role in DNA repair (Cairns- deLucia mutant was UV-sensitive)
- its role in DNA replication is to remove primers and fill in the gaps left behind
- for this it needs the nick-translation activity 9

37. The DNA Polymerase Family
A total of 5 different DNAPs have been reported in E. coli
DNAP I: functions in repair and replication
DNAP II: functions in DNA repair (proven in 1999)
DNAP III: principal DNA replication enzyme
DNAP IV: functions in DNA repair (discovered in 1999)
DNAP V: functions in DNA repair (discovered in 1999) 9

38. The "real" replicative polymerase in E. coli
DNA Polymerase III
The "real" replicative polymerase in E. coli
It’s fast: up to 1,000 dNTPs added/sec/enzyme
It’s highly processive: >500,000 dNTPs added before dissociating
It’s accurate: makes 1 error in 107 dNTPs added, with proofreading, this gives a final error rate of 1 in 1010 overall. 9