1. Chapter 10 Replication of DNA
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2. Chapter Outline Basic Features of DNA Replication In Vivo
DNA Replication in Prokaryotes
Unique Aspects of Eukaryotic DNA Replication
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3. Basic Features of DNA Replication In Vivo
DNA replication occurs semiconservatively, is initiated at unique origins, and usually proceeds bidirectionally from each origin of replication.
Synthesis of DNA (RNA,proteins):
1-initiation, 2-extension/elongation, 3-termiantion.
DNA polymerase (protein-enzyme)-essential for conservation of any species
3,000/30,000 nucleotides per minutes
One mistake per billion of nucleotides
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4. DNA Replication is Semiconservative
MODEL
Each strand serves as a template
Complementary base pairing determines the sequence of the new strand
Each strand of the parental helix is conserved
Semiconservative=half conserve
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5. Possible Models of DNA Replication
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6. CsCl Equilibrium Density-Gradient Centrifugation
Bacteria growing with 15N and 14N (normal)
Density:
GsCl
~1.7 g/cm3
DNA
~1.710 g/cm3 with 14N
~1.724 g/cm3 with 15N
Bacteria with 15N-parental
(several periods of time)
Bacteria with 14N-daugther
Centrifugation:
process involving the centrifugal force for the
sedimentation of particles and/or molecules
[revolutions per minute (RPM)]
gravitational force to cause precipitation/sedimentation
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10. The Meselson-Stahl Experiment: DNA Replication in E
The Meselson-Stahl Experiment: DNA Replication in E. coli is Semiconservative
Bacteria growing with 15N for several generations
Change medium and add 14N
--one generation
--two generations
--three generations
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15. Semiconservative Replication in Eukaryotes
Autoradiography: A technique using
X- ray film to visualize molecules or
fragments of molecules that have been
radioactively labeled
1H-Thymidine (normal)
3H-Thymidine
Autoradiography
3H=tritium
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16. C-metaphase: Colchicine-metaphase:
3H-Thymidine
1H-Thymidine
C-metaphase: Colchicine-metaphase:
Colchincine: is a toxic natural product and secondary metabolite
and it inhibits microtubule polymerization by binding to tubulin.
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17. The Origin of Replication in E. coli
Replicon: is a sequence of DNA at which DNA replication is initiated on a chromosome, plasmid or virus.
-OriC (245 bp)
-AT-rich region (replication bubble)
-13-mer and 9-mer tandem
N: any nucleotide
Eukaryotic:
ARS
(Autonomously Replicating sequences)
AT-rich region 11bp
Mer=repeating unit=parts
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18. Bidirectional Replication of the Circular E. coli Chromosome
-Circular DNA (double strand DNA)
--Unwind (access and single strand DNA)
--Simultaneous semiconservative replication
--Swivel (point of break) Topoisomerases
--Y-shape structure=replication fork
Topoisomerases: are enzymes that regulate the overwinding or underwinding of DNA.
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19. Visualization of Replication in E. coli
3H-Thymidine
Autoradiography
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20. Visualization of Replication in E. coli
3H-Thymidine
Autoradiography
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21. Bidirectional Replication: The Phage Chromosome
-Small bacterial virus
Single stranded DNA (12 bp)
Cohesive/sticky and complementary ends
DNA ligase (replication, repair and recombination)
Linear
Circular
replication
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22. Replication is Bidirectional
1-AT- and CG -rich regions
Native Denature
2-Bubbles
3-DNA polymerase access
100°C
pH~11
~10 min
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23. Replication is Bidirectional
Both branch points ( Y shape) are replication forks
Replication fork: junction where the double-stranded DNA splits apart
(or unzipped) into 2 single strands.
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24. Bidirectional Replication of Phage T7
--eye structure
replication forks
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25. DNA replicates by a semiconservative mechanism: as the two complementary strands of a parental double helix unwind and separate, each serves as a template for the synthesis of a new complementary strand.
The hydrogen-bonding potentials of the bases in the template strands specify complementary base sequences in the nascent DNA strands.
Replication is initiated at unique origins and usually proceeds bidirectionally from each origin.
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26. DNA Replication in Prokaryotes
DNA replication is a complex process, requiring the concerted action of a large number of proteins
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27. DNA Polymerases and DNA Synthesis In Vitro
Much of what we know about DNA synthesis was deduced from in vitro studies.
DNA Polymerase I
Single polypeptides
5’ to 3’
Triphosphate [dATP]
MgCl2
Free 3’OH group of the DNA strands
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28. Continuous vs discontinuous--leading and lagging strands
Replicating fork
Bacteriophage T4
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29. Small fragments to big fragments
1,000 to 2,000 bp
Okazaki fragments
10,000 to 200,000 bp
Small fragments to big fragments
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30. Continuous vs discontinuous--leading and lagging strands
Replicating fork
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31. © John Wiley & Sons, Inc.

32. Prepriming at oriC in E. coli
--Replication bubbles
Self aggregation
DNA helicase:
it separates two annealed nucleic acid
Strands.
Why?
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33. RNA Primers are Used to Initiate DNA Synthesis
DNA primase: short RNA primer
RNA/DNA hybrid
(unstable ?)
Perfect conditions for
DNA polymerases to work
(free 3’OH)
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34. DNA Polymerase I: 5'3' Polymerase Activity
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35. DNA Polymerase I: 5'3' Exonuclease Activity
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36. DNA Polymerase I: 3'5' Exonuclease Activity
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37. © John Wiley & Sons, Inc.

38. DNA Helicase Unwinds the Parental Double Helix
One of the most important event during DNA replication
Why?
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39. Single-Strand DNA Binding (SSB) Protein
Access to DNA polymerase
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40. Supercoiling of Unwound DNA
DNA Topoisomerases I: produce single transient breaks of DNA and remove supercoiling
It blocks DNA replication
DNA Topoisomerases II: produce double transient breaks of
DNA and negative supercoiling (DNA gyrase)
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41. DNA Topoisomerase I Produces Single-Strand Breaks in DNA
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42. Requirements of DNA Polymerases
Primer DNA with free 3'-OH
Template DNA to specify the sequence of the new strand
Substrates: dNTPs
Mg2+
Reaction: nucleophilic attack
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43. DNA Polymerase III is the True DNA Replicase of E. coli
--a 900 KDa multimeric protein
--Dimers
--Holoenzymes
--High fidelity (error ~1 in a 1 x 1012)
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44. Proofreading mechanism
Subunits----Prokaryotes
 Subunits----Eukaryotes
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45. The Replication Apparatus in E. coli
Primosome:
Initiation of Okazaki fragment during lagging strand
DNA primase and DNA helicase
DnaB and C proteins
Require ATP
DNA helicase:unwinds DNA
DNA primase: synthesis of RNA
Topoisomerase: transient DNA breaks
DNA polymerase III: extend the RNA primers (deoyxribonucleotide). It is holoenzymes
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46. DNA Replication Synthesis of the leading strand is continuous.
Synthesis of the lagging strand is discontinuous. The new DNA is synthesized in short segments (Okazaki fragment) that are later joined together.
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47. The E. coli Replisome Replisome: complete replication apparatus
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48. Rolling-Circle Replication
Replication’s Models-
O-shape
Eye-shape
Y-shape
Rolling-circle (viruses, bacteria , amphibians)
_______________________________________
1- Nick by specific endonucleases
2-parental DNA is intact and functions as template
3-DNA polymerase 5’ to 3’
4- displacement of one of the DNA strand
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49. DNA replication is complex, requiring the participation of a large number of proteins.
DNA synthesis is continuous on the progeny strand that is being extended in the overall 5'3' direction, but is discontinuous on the strand growing in the overall 3'5' direction.
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50. DNA synthesis is catalyzed by enzymes called DNA polymerases.
New DNA chains are initiated by short RNA primers synthesized by DNA primase.
DNA synthesis is catalyzed by enzymes called DNA polymerases.
All DNA polymerases require a primer strand, which is extended, and a template strand, which is copied.
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51. All DNA polymerases have an absolute requirement for a free 3’-OH on the primer strand, and all DNA synthesis occurs in the 5’ to 3’ direction.
The 3’ to 5’ exonuclease activities of DNA polymerases proofread nascent strands as they are synthesized, removing any mispaired (match) nucleotides at the 3’ termini of primer strands.
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52. The enzymes and DNA-binding proteins involved in replication assembled into a replisome at each replication fork and act in concert as the fork moves along the parental DNA molecule.
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53. Unique Aspects of Eukaryotic Chromosome Replication
Although the main features of DNA replication are the same in all organisms, some processes occur only in eukaryotes.
Viruses and E.coli
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54. DNA Replication in Eukaryotes
Shorter RNA primers and Okazaki fragments
DNA replication only during S phase
(bacteria will duplicate DNA only in a rich environment)
Multiple origins of replication
(bacteria shows one origins of replication)
Nucleosomes
(nucleosomes are not present in bacteria)
Telomeres
(telomeres are not present in bacteria)
Cell Cycle
--check points
----S phase
----Mitosis
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55. Bidirectional Replication from Multiple Origins in Eukaryotes
Pulse chase experiments with 3H-thymidine
---Origins of replication
---Large number of replicons
(1 vs ~1x105)
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56. Replicon: segment of DNA containing one Origin (O) and Two termini (T)
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57. The Eukaryotic Replisome
SV40 virus: DNA virus (histones)
Bacteria replication
--unwind parental DNA (without histones)
----DNA helicase
----Topoisomerase
----Single -strand DNA binding protein
----DNA polymerase III
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58. Eukaryotic Replication Proteins
----parental DNA (with histones)
----Polymerases ()
Pol initiation of replication (origins)
priming of Okazaki fragment
complex with DNA primase
Pol synthesis of lagging strand
 Pol synthesis of leading strand
----accessories proteins: PCNA and Rf-C
(sliding clamp)
----Pol  have exonuclease activity ( 3’to 5”)
=proofreading
----Other Pols (pie, lambda, phi, rho, and mu)
do not have exonuclease activity ( 5’to 3”)
----Ribonulceases H1 and FEN-1
Produce the
RNA/DNA chain
Proliferating Cell Nuclear Antigen: PCNA
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59. Nucleosome Spacing in Replicating Chromatin
Assembly and disassembly of nucleosomes
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60. Chromatin can have alternative states
Inactive--DNA/histones
Active--Polymerase/TFs
Polymaerase/TFs
NO TRANSCRIPTION
HISTONES
TRANSCRIPTION
“The addition of either TFs or nucleosomes may form stable structures
that can not be changed by modifying the equilibrium with free
components”
How is the chromatin structure regulated?

61. Chromatin remodeling
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62. The Telomere “extension” Problem
DNA polymerase can not replicate the terminal DNA
---too big
---not enough space ( 3’-OH, primer)
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63. Telomerase (Reverse Transcriptase) G-rich telomere sequence 5’ to 3’
Aging (early aging….progerias)
Immortality:
Cancer and Normal cells
Senescence:
normal diploid cells cease to divide,
(about 45 to 50 cell divisions).
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64. Telomere Length and Aging
Shorter telomeres are associated with cellular senescence and death.
Diseases causing premature aging are associated with short telomeres.
Apoptosis (self-destruction):
programmed cell death
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66. Telomeres Are Essential for Survival
Figure 28.32

67. Dna polymerases classification as follows:
Prokaryotic DNA polymerases
Pol I to V
Eukaryotic DNA polymerases
Pol  theta, pie, lambda, phi, rho, and mu.
Based on sequence homology
A, B, C, D, X, Y, and RT

bacterial
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68. Since the parental double helix must rotate 360° to unwind each gyre of the helix,
during the semi-conservative replication of the bacterial chromosome, some kind of “swivel” must exist.
What do geneticists now know that the required swivel is?
a) Topoisomerase
b) Helicase
c) A transient single-strand break produced by the action of topoisomerases
d) A transient single-strand break produced by the action of helicases
e) A transient single-strand break produced by the action of Ligase
In the E. coli chromosome the origin of replication, called oriC, is characterized as being rich in:
a) A-G base pairs
b) A-C base pairs
c) C-G base pairs
d) C-T base pairs
e) None of the above

69. Telomere length has not been correlated with:
a) Aging
b) Sex determination
c) Progeria
d) Cancer
e) All of these