1. REPLICATION
Chapter 7

2. The Problem DNA is maintained in a compressed, supercoiled state.
BUT, basis of replication is the formation of strands based on specific bases pairing with their complementary bases.
 Before DNA can be replicated it must be made accessible, i.e., it must be unwound

3. Models of Replication THREE HYPOTHESES 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
THREE HYPOTHESES FOR DNA REPLICATION

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

5. PREDICTED DENSITIES OF NEWLY REPLICATED DNA
MOLECULES ACCORDING
TO THE THREE HYPOTHESES
ABOUT DNA REPLICATION
Density Gradient Centrifugation can be used to address the question.
WHAT RESULTS ARE PREDICTED?

6. Meselson and Stahl Conclusion: Semi-conservative replication of DNA

7. Replication as 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.

8. Extending the Chain dNTPs are added individually
Sequence determined by pairing with template strand
DNA has only one phosphate between bases, so why use dNTPs?
Deoxyribonucleoside triphosphates are the building blocks of DNA. However, a complete polynucleotide strand of DNA has only one phosphate group and that through this phosphate group each nucleotide is attached to the next. Why then is the substrate a triphosphate instead of just a monophosphate? The answer to this question lies in the chemistry underlying the addition of nucleotides to a growing daughter strand of DNA.

9. Extending the Chain
While each nucleotide added to a growing DNA chain lacks an -OH group at its 2' position, it retains its 3' -OH. This hydroxyl group is used to attack the alpha phosphate group of an incoming nucleoside triphosphate. In the attack, the 3' -OH replaces the beta and gamma phosphates that are ejected from the complex as a pyrophosphate molecule. The result is the formation of the phosphodiester bond between the growing daughter strand and the next nucleotide. The 3' -OH of the newly added nucleotide is now exposed on the end of the growing chain and can attack the next nucleotide in the same way.
The figure above presents a simplified schematic of a growing polynucleotide chain. The lines represent the ribose sugar with one 3' -OH branching from it. Each p represents a phosphate group. This figure illustrates a number of key points of DNA replication. First, we see that the parent strand is oriented in the 3' to 5' direction. Second, each new nucleotide added to the growing daughter strand is complementary to the nucleotide on the parent strand that is across from it and a bond forms between them. Finally, we see how the 3' -OH group displaces the two outermost phosphate groups of an incoming nucleotide in order to add it to the growing chain.
The Driving Force of the Addition Reaction
Each incoming nucleotide supplies the energy for its addition in the high-energy bond between the beta and gamma phosphates that are ejected upon addition. It is not the release of the pyrophosphate that drives the reaction, but rather the subsequent hydrolysis that takes place. A much larger amount of energy is released when the two are split by inorganic pyrophosphatase to yield two phosphates. Total G~ -7 kcal/mol., offsetting the loss in entropy when the nucleotide is added (~0.5 kcal/mol).

10. DNA Synthesis
3’-OH nucleophilic attack on alpha phosphate of incoming dNTP
nucleotide gets positioned through H- bonding with template
- 3’-OH nucleophilic attack on alpha phosphate of incoming dNTP.
loss of entropy; not much gain in bond-energy
reaction is driven by removal and splitting of pyrophosphate
because of requirement for 3’-OH and 5’ dNTP substrate, DNA polymerase can only catalyze reaction in the 5’ 3’ direction (direction of new strand!)
removal and splitting of pyrophosphate by inorganic pyrophosphatase
2 phosphates

11. Chain Elongation in the 5’  3’ direction
2. Reaction
Template Growing strand: 5' 3'
strand
Pol I
3. Reaction requirements
a. DNA template
b. 3 - OH primer; can be RNA or DNA
c. dNTP’s and Mg2+
Primer
Incoming dNTP -
Nucleophilic attack
of primer strand 3’ OH
on the phosphate of
the dNTP

12. Semi-discontinuous Replication
All known DNA pols work in a 5’>>3’ direction
Solution?
Okazaki fragments

13. Okazaki Experiment

14. Continuous synthesis
Discontinuous synthesis
DNA replication is semi-discontinuous

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

16. DNA Replication-Prokaryotes
DNA replication is semiconservative. the helix must be unwound.
Most naturally occurring DNA is slightly negatively supercoiled.
Torsional strain must be released
Replication induces positive supercoiling
Torsional strain must be released, again.
SOLUTION: Topoisomerases

17. The Problem of Overwinding
Relaxation of closed circular replicating DNA by topoisomerases. The problem: As Replication of closed circular DNA proceeds, an overwound region (in the unreplicated portion) is formed as a result of unwinding on the other side of the molecule.

18. Topoisomerase Type I Precedes replicating DNA Mechanism
Makes a cut in one strand, passes other strand through it. Seals gap.
Result: induces positive supercoiling as strands are separated, allowing replication machinery to proceed.

19. Gyrase--A Type II Topoisomerase
Introduces negative supercoils
Cuts both strands
Section located away from actual cut is then passed through cut site.

20. Helicase Operates in replication fork
Separates strands to allow DNA Pol to function on single strands.
Involves breaking H-bonds and hydrophobic interactions
Requires ATP
Operates in replication fork
Separates strands to allow DNA Pol to function on single strands.
Translocate along single strain in 5’->3’ or 3’-> 5’ direction by hydrolyzing ATP

21. Initiation of Replication
Replicaion initiated at specific sites: Origin of Replication (ori)
Two Types of initiation:
De novo –Synthesis initiated with RNA primers. Most common.
Covalent extension—synthesis of new strand as an extension of an old strand (“Rolling Circle”)

22. De novo Initiation Binding to Ori C by DnaA protein Opens Strands
Replication proceeds bidirectionally
Binding to Ori C by DnaA protein. Is a 50 kD protein that recognizes and interacts with specific repeated sequences (9-mers) in the oriC region. This recognition sequence is a set of 9 nt, repeated 4 times.
Several DnaA proteins come together to form a complex w/~150 bp DNA This formation denatures a region of the DNA (characterized by repeating 13 mers [AT rich region]) adjacent to the initial complex.
Forms an open complex.
it is now open and accessible to the other replications proteins to form the replication fork.
Replication proceeds bidirectionally by extension of RNA primers

23. Unwinding the DNA by Helicase (DnaB protein)
Uses ATP to separate the DNA strands
At least 4 helicases have been identified in E. coli.
How was DnaB identified as the helicase necessary for replication?
NOTE: Mutation in such an essential gene would be lethal.
Solution?
Conditional mutants
Uses ATP to separate the DNA strands
At least 4 helicases have been identified in E. coli.:
rep helicase
helicase II
Helicase III
DnaB protein
How was DnaB identified as the helicase necessary for replication?
NOTE: Mutation in such an essential gene would be lethal, undetectable.
Solution?
Conditional mutants—i.e., mutation expressed only under certain conditions, ex. Elevated T.
Jab isolated mutants that ceased DNA
One had a mutation in the DnaB gene.

24. Liebowitz Experiment
What would you expect if the substrates are separated by electrophoresis after treatment with a helicase?
Helicase Assay
Does DnaB Encode Rplication Helicase?
Used single stranded DNA encoded by M-13 (a phage)
Made a labeled 1.06 fragment complementary to the phage DNA
Tested DnaB protein, DnaG protein, an single stranded DNA binding proteins

25. Liebowitz Assay--Results
What do these results indicate?
ALTHOUGH PRIMASE (DnaG) AND SINGLE- STRAND BINDING PROTEIN (SSB) BOTH STIMULATE DNA HELICASE (DnaB), NEITHER HAVE HELICASE ACTIVITY OF THEIR OWN
ALTHOUGH PRIMASE (DnaG) AND SINGLE- STRAND BINDING PROTEIN (SSB) BOTH STIMULATE DNA HELICASE (DnaB), NEITHER HAVE HELICASE ACTIVITY OF THEIR OWN

26. Single Stranded DNA Binding Proteins (SSB)
Maintain strand separation once helicase separates strands
Not only separate and protect ssDNA, also stimulates binding by DNA pol (too much SSB inhibits DNA synthesis)
Strand growth proceeds 5’>>3’
Maintain strand separation once helicase separates strands
Not only separate and protect ssDNA, also stimulates binding by DNA pol
Strand growth proceeds 5’>>3’

27. Replication: The Overview
Requirements:
Deoxyribonucleotides
DNA template
DNA Polymerase
5 DNA pols in E. coli
5 DNA pols in mammals
Primer
Proofreading
High Fidelity DNA Replication
Expected error rate=1 mistake/103 nt
Error rate= 1 mistake/109 nucleotides
Afforded by complementary base pairing and proof-reading capability of DNA polymerase

28. Overview of replication
Overview of replication. We will start with a discussion of the primary polymerizing enzymes (pol I and pol II)

29. DNA pol I First DNA pol discovered. Proteolysis yields 2 chains
Larger Chain (Klenow Fragment) 68 kd
C-terminal 2/3rd. 5’>>3’ polymerizing activity
N-terminal 1/3rd. 3’>>5’ exonuclease activity
Smaller chain: 5’>>3 exonucleolytic activity
nt removal 5’>>3’
Can remove >1 nt
Can remove deoxyribos or ribos
A very talented enzyme.
First DNA pol discovered.
Several activities, but occurs as a monomer despite multiple functions. Various functions in separate domains.
Proteolysis yields 2 chains
Larger Chain (Klenow Fragment) 68 kd
C-terminal 2/3rds---5’>>3’ polymerizing activity
N-terminal 1/3rd. 3’>>5’ exonuclease activity
Smaller chain: 5’>>3 exonucleolytic activity
nt removal 5’>>3’
Can remove >1 nt
Can remove deoxyribos or ribos
NOTE: can be active at a nick in the strand as long as there is a 5’ phosphate
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

30. Nick translation Requires 5’-3’ activity of DNA pol I Steps
At a nick (free 3’ OH) in the DNA the DNA pol I binds and digests nucleotides in a 5’-3’ direction
The DNA polymerase activity synthesizes a new DNA strand
A nick remains as the DNA pol I dissociates from the ds DNA.
The nick is closed via DNA ligase
Requires 5’-3’ activity of DNA pol I
Steps
At a nick (free 3’ OH) in the DNA the DNA pol I binds and digests nucleotides in a 5’-3’ direction
The DNA polymerase activity synthesizes a new DNA strand
A nick remains as the DNA pol I dissociates from the ds DNA.
The nick is closed via DNA ligase
Uses:
removal of RNA primers
DNA repair
Great for DNA labeling with radioactive dNTPs
Source: Lehninger pg. 940