1. PowerPoint Presentation Materials to accompany
Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 11
DNA REPLICATION
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2. INTRODUCTION
DNA replication is the process by which the genetic material is copied
It occurs
very quickly,
very accurately and
at the appropriate time in the life of the cell
This chapter examines how!
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4. 11.1 STRUCTURAL OVERVIEW OF DNA REPLICATION
DNA replication relies on the complementarity of DNA strands
The AT/GC rule or Chargaff’s rule
The process can be summarized as such
The two DNA strands come apart
Each serves as a template strand for the synthesis of new strands
The two newly-made strands = daughter strands
The two original ones = parental strands
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5. Identical base sequences
Figure 11.1
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6. Experiment 11A: Which Model of DNA Replication is Correct?
In the late 1950s, three different mechanisms were proposed for the replication of DNA
Conservative model
Both parental strands stay together after DNA replication
Semiconservative model
The double-stranded DNA contains one parental and one daughter strand following replication
Dispersive model
Parental and daughter DNA are interspersed in both strands following replication
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7. Figure 11.2
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8. 11.2 BACTERIAL DNA REPLICATION
DNA synthesis begins at a site termed the origin of replication
Each bacterial chromosome has only one
Synthesis of DNA proceeds bidirectionally around the bacterial chromosome
The replication forks eventually meet at the opposite side of the bacterial chromosome
This ends replication
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9. Figure 11.4
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10. Initiation of Replication
The origin of replication in E. coli is termed oriC
origin of Chromosomal replication
Three types of DNA sequences in oriC are functionally significant
AT-rich region
DnaA boxes
GATC methylation sites
Refer to Figure 11.5
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11. Figure 11.5
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12. 11-17 Figure 11.6 Other proteins such as HU and IHF also bind.
This causes the region to wrap around the DnaA proteins and separates the AT-rich region
DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences
This binding stimulates the cooperative binding of an additional 20 to 40 DnaA proteins to form a large complex
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13. Travels along the DNA in the 5’ to 3’ direction
Figure 11.6
Composed of six subunits
Travels along the DNA in the 5’ to 3’ direction
Uses energy from ATP
Bidirectional replication
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14. This generates positive supercoiling ahead of each replication fork
DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them
This generates positive supercoiling ahead of each replication fork
DNA gyrase travels ahead of the helicase and alleviates these supercoils
Single-strand binding proteins bind to the separated DNA strands to keep them apart
Then short (10 to 12 nucleotides) RNA primers are synthesized by DNA primase
These short RNA strands start, or prime, DNA synthesis
The leading strand has a single primer, the lagging strand needs multiple primers
They are later removed and replaced with DNA
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15. 11-20 Figure 11.7 LEADING STRAND LOGGING STRAND
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16. 11-21
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17. DNA Polymerases
DNA polymerases are the enzymes that catalyze the attachment of nucleotides to make new DNA
In E. coli there are five proteins with polymerase activity
DNA pol I, II, III, IV and V
DNA pol I and III
Normal replication
DNA pol II, IV and V
DNA repair and replication of damaged DNA
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18. DNA Polymerases DNA pol I DNA pol III Composed of a single polypeptide
Removes the RNA primers and replaces them with DNA
DNA pol III
Responsible for most of the DNA replication
Composed of 10 different subunits (Table 11.2)
The a subunit synthesizes DNA
The other 9 fulfill other functions
The complex of all 10 is referred to as the DNA pol III holoenzyme
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20. 11-24

23. Bacterial DNA polymerases may vary in their subunit composition
However, they have the same type of catalytic subunit
Structure resembles a human right hand
Template DNA thread through the palm;
Thumb and fingers wrapped around the DNA
Figure 11.8
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24. 11-26 Figure 11.9 Unusual features of DNA polymerase function
DNA polymerases cannot initiate DNA synthesis
Problem is overcome by the RNA primers synthesized by primase
Problem is overcome by synthesizing the 3’ to 5’ strands in small fragments
DNA polymerases can attach nucleotides only in the 5’ to 3’ direction
Unusual features of DNA polymerase function
Figure 11.9
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27. The two new daughter strands are synthesized in different ways
Leading strand
One RNA primer is made at the origin
DNA pol III attaches nucleotides in a 5’ to 3’ direction as it slides toward the opening of the replication fork
Lagging strand
Synthesis is also in the 5’ to 3’ direction
However it occurs away from the replication fork
Many RNA primers are required
DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each)
These are termed Okazaki fragments after their discoverers
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28. DNA pol I removes the RNA primers and fills the resulting gap with DNA
It uses its 5’ to 3’ exonuclease activity to digest the RNA
and its 5’ to 3’ polymerase activity to replace it with DNA
After the gap is filled a covalent bond is still missing
DNA ligase catalyzes a phosphodiester bond
Thereby connecting the DNA fragments
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29. Figure 11.10
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30. DNA polymerase III synthesizes base pairs at a rate of around 1000 nucleotides per second

31. Termination of Replication
Opposite to oriC is a pair of termination sequences called ter sequences
These are designated T1 and T2
T1 stops counterclockwise forks, T2 stops clockwise
The protein tus (termination utilization substance) binds to these sequences
It can then stop the movement of the replication forks
Refer to Figure 11.12
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32. 11-35 Figure 11.12 Allows the advancement of the CW-moving fork
Prevents the movement of the CCW-moving fork
Allows the advancement of the CCW-moving fork
Prevents the movement of the CW-moving fork
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33. Termination of Replication
DNA replication ends when oppositely advancing forks meet (usually at T1 or T2)
Finally DNA ligase covalently links all four DNA strands
DNA replication often results in two intertwined molecules
Intertwined circular molecules are termed catenanes
These are separated by the action of topoisomerases
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34. Catalyzed by DNA topoisomerases
Figure 11.13
Catenanes
Catalyzed by DNA topoisomerases
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35. DNA Replication Complexes
DNA helicase and primase are physically bound to each other to form a complex called the primosome
This complex leads the way at the replication fork
The primosome is physically associated with the DNA polymerase holoenzyme forming the replisome
Figure provides a three-dimensional view of DNA replication
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36. Figure 11.14
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37. Proofreading Mechanisms
DNA replication exhibits a high degree of fidelity
Mistakes during the process are extremely rare
DNA pol III makes only one mistake per 108 bases made
There are several reasons why fidelity is high
1. Instability of mismatched pairs
2. Configuration of the DNA polymerase active site
3. Proofreading function of DNA polymerase
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38. Proofreading Mechanisms
1. Instability of mismatched pairs
Complementary base pairs have much higher stability than mismatched pairs
This feature only accounts for part of the fidelity
It has an error rate of 1 per 1,000 nucleotides
2. Configuration of the DNA polymerase active site
DNA polymerase is unlikely to catalyze bond formation between mismatched pairs
This induced-fit phenomenon decreases the error rate to a range of 1 in 100,000 to 1 million
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39. Proofreading Mechanisms
3. Proofreading function of DNA polymerase
DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand
The enzyme uses its 3’ to 5’ exonuclease activity to remove the incorrect nucleotide
It then changes direction and resumes DNA synthesis in the 5’ to 3’ direction
Refer to figure 11.15
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40. 11-45 Figure 11.15 A schematic drawing of proofreading
Site where DNA backbone is cut
A schematic drawing of proofreading
Figure 11.15
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41. Bacterial DNA Replication is Coordinated with Cell Division
Bacterial cells can divide into two daughter cells at an amazing rate
E. coli  20 to 30 minutes
Therefore it is critical that DNA replication take place only when a cell is about to divide
Bacterial cells regulate the DNA replication process by controlling the initiation of replication at oriC
E. coli does this via two different mechanisms
Refer to Figures and 11.17
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42. The amount of DnaA protein provides a way to regulate DNA replication
Figure 11.16
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43. 11-48 Figure 11.17 DNA adenine methyltransferase
Recognizes the 5’ – GATC – 3’ sequence and attaches a methyl group onto the adenine
DNA adenine methyltransferase
Methylation of GATC sites in oriC
Figure 11.17
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44. 11-49 Figure 11.17 Methylation of GATC sites in oriC
Before replication, GATC sites are methylated on both strands
This full methylation facilitates initiation of DNA replication
Following replication, GATC sites are not methylated on the daughter strands
This half-methylation does not efficiently initiate replication
Several minutes will pass before Dam methylase will methylate the GATC sites in the daughter strands
Methylation of GATC sites in oriC
Figure 11.17
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45. 11.3 EUKARYOTIC DNA REPLICATION
Eukaryotic DNA replication is not as well understood as bacterial replication
The two processes do have extensive similarities,
The bacterial enzymes described in Table 11.1 have also been found in eukaryotes
Nevertheless, DNA replication in eukaryotes is more complex
Large linear chromosomes
Tight packaging within nucleosomes
More complicated cell cycle regulation
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46. Multiple Origins of Replication
Eukaryotes have long linear chromosomes
They therefore require multiple origins of replication
To ensure that the DNA can be replicated in a reasonable time
In 1968, Huberman and Riggs provided evidence for the multiple origins of replication
Refer to Figure 11.20
DNA replication proceeds bidirectionally from many origins of replication
Refer to Figure 11.21
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47. Figure 11.21
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50. Multiple Origins of Replication
The origins of replication found in eukaryotes have some similarities to those of bacteria
Origins of replication in Saccharomyces cerevisiae are termed ARS elements (Autonomously Replicating Sequence)
They are bp in length
They have a high percentage of A and T
They have three or four copies of a specific sequence
Similar to the bacterial DnaA boxes
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51. Multiple Origins of Replication
Replication begins with assembly of the prereplication complex (preRC)
Consist of 14 different proteins. 6 of them called origin replication complex.
An important part of this is the Origin recognition complex (ORC)
A six-subunit complex that acts as the initiator of eukaryotic DNA replication
DNA replication at the origin begins with the binding of ORC, which usually occurs during G1 phase.
Other preRC proteins bind including MCM helicase
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52. MCM is an acronym for minichromosome maintenance.

53. Binding of MCM completes DNA replication licensing
The origin is capable of initiating DNA synthesis
only those origins with MCM helicase can initiate DNA synthesis
Binding of at least 22 additional proteins is required to initiate synthesis during S phase

54. Bénédicte Recolin et al 2014

55. Eukaryotes Contain Several Different DNA Polymerases
Mammalian cells contain well over a dozen different DNA polymerases
Refer to Table 11.4
Four: alpha (a), delta (d), epsilon (e) and gamma (g) have the primary function of replicating DNA
a, d and e  Nuclear DNA
g  Mitochondrial DNA
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57. DNA pol a is the only polymerase to associate with primase
The DNA pol a/primase complex synthesizes a short RNA-DNA hybrid
10 RNA nucleotides followed by 20 to 30 DNA nucleotides
This is used by DNA pol d or e for the processive elongation of the leading and lagging strands
Current evidence suggests a greater role for DNA pol d
The exchange of DNA pol a for d or e is called a polymerase switch
It occurs only after the RNA-DNA hybrid is made
Refer to Figure 11.22
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58. Figure 11.22
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59. DNA polymerases also play a role in DNA repair
DNA pol b is not involved in DNA replication
It plays a role in base-excision repair
Removal of incorrect bases from damaged DNA
Recently, more DNA polymerases have been identified
Lesion-replicating polymerases
Involved in the replication of damaged DNA
They can synthesize a complementary strand over the abnormal region
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60. Telomeres and DNA Replication
Linear eukaryotic chromosomes have telomeres at both ends
The term telomere refers to the complex of telomeric DNA sequences and bound proteins
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61. Telomeric sequences consist of
Moderately repetitive tandem arrays TTAGG
3’ overhang that is nucleotides long
Figure 11.23
Telomeric sequences typically consist of
Several guanine nucleotides
Often many thymine nucleotides
Refer to Table 11.5
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63. DNA polymerases possess two unusual features
1. They synthesize DNA only in the 5’ to 3’ direction
2. They cannot initiate DNA synthesis
These two features pose a problem at the 3’ end of linear chromosomes-the end of the strand cannot be replicated!
Figure 11.24
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64. Therefore if this problem is not solved
The linear chromosome becomes progressively shorter with each round of DNA replication
Indeed, the cell solves this problem by adding DNA sequences to the ends of telomeres
This requires a specialized mechanism catalyzed by the enzyme telomerase
Telomerase contains protein and RNA
The RNA is complementary to the DNA sequence found in the telomeric repeat
This allows the telomerase to bind to the 3’ overhang
The lengthening mechanism is outlined in Figure 11.25
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65. Step 1 = Binding
The binding-polymerization-translocation cycle can occurs many times
Step 2 = Polymerization
This greatly lengthens one of the strands
Step 3 = Translocation
The end is now lengthened
Figure 11.25
RNA primer
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