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
The original DNA strands are used as templates for the synthesis of new strands
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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3. 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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4. Identical base sequences
Figure 11.1
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5. 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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6. Figure 11.2
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7. Their experiment can be summarized as such
In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models
They found a way to experimentally distinguish between daughter and parental strands
Their experiment can be summarized as such
Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen) for many generations
The population of cells had heavy-labeled DNA
Switch E. coli to medium containing only 14N (a light isotope of Nitrogen)
Collect sample of cells after various times
Analyze the density of the DNA by centrifugation using a CsCl gradient
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8. Testing the Hypothesis
This experiment aims to determine which of the three models of DNA replication is correct
Testing the Hypothesis
Refer to Figure 11.3
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9. Figure 11.3
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10. DNA
Figure 11.3
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11. The Data
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12. Interpreting the Data 11-12 After one generation, DNA is “half-heavy”
After ~ two generations, DNA is of two types: “light” and “half-heavy”
This is consistent with both semi-conservative and dispersive models
This is consistent with only the semi-conservative model
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13. 11.2 BACTERIAL DNA REPLICATION
Figure 11.4 presents an overview of the process of bacterial chromosome 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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14. Figure 11.4
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15. 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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16. Figure 11.5
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17. 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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18. 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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19. 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
They are later removed and replaced with DNA
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20. 11-20 Figure 11.7 Keep the parental strands apart
Breaks the hydrogen bonds between the two strands
Alleviates supercoiling
Synthesizes an RNA primer
Figure 11.7
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21. 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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22. 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
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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23. 11-23

24. 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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25. 11-25 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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26. 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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27. 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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28. 11-28 Figure 11.7 Keep the parental strands apart
Breaks the hydrogen bonds between the two strands
Alleviates supercoiling
Keep the parental strands apart
Synthesizes an RNA primer
Synthesizes daughter DNA strands
III
Covalently links DNA fragments together
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29. The Reaction of DNA Polymerase
DNA polymerases catalyzes a phosphodiester bond between the
Innermost phosphate group of the incoming deoxynucleoside triphosphate
AND
3’-OH of the sugar of the previous deoxynucleotide
In the process, the last two phosphates of the incoming nucleotide are released
In the form of pyrophosphate (PPi)
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30. Figure 11.10
Innermost phosphate
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31. DNA Polymerase III is a Processive Enzyme
DNA polymerase III remains attached to the template as it is synthesizing the daughter strand
This processive feature is due to several different subunits in the DNA pol III holoenzyme
b subunit is in the shape of a ring
It is termed the clamp protein
g subunit is needed for b to initially clamp onto the DNA
It is termed the clamp-loader protein
d, d’ and y subunits are needed for the optimal function of the a and b subunits
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32. DNA Polymerase III is a Processive Enzyme
The effect of processivity is quite remarkable
In the absence of the b subunit
DNA pol III falls off the DNA template after a few dozen nucleotides have been polymerized
Its rate is ~ 20 nucleotides per second
In the presence of the b subunit
DNA pol III stays on the DNA template long enough to polymerize up to 50,000 nucleotides
Its rate is ~ 750 nucleotides per second
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33. Termination of Replication
Opposite to oriC is a pair of termination sequences called ter sequences
These are designated T1 and T2
The protein tus (termination utilization substance) binds to these sequences
It can then stop the movement of the replication forks
Refer to Figure 11.11
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34. Figure 11.11
Allows the advancement of the CW-moving fork
Prevents the movement of the CCW-moving fork
(T1)
(T2)
Allows the advancement of the CCW-moving fork
Prevents the movement of the CW-moving fork
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35. 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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36. Catalyzed by DNA topoisomerases
Figure 11.12
Catenanes
Catalyzed by DNA topoisomerases
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37. QUICK REVIEW
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38. 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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39. Figure 11.13
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40. DNA Replication Complexes
Two DNA pol III proteins act in concert to replicate both the leading and lagging strands
The two proteins form a dimeric DNA polymerase that moves as a unit toward the replication fork
DNA polymerases can only synthesize DNA in the 5’ to 3’ direction
So synthesis of the leading strand is continuous
And that of the lagging strand is discontinuous
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41. DNA Replication Complexes
Lagging strand synthesis is summarized as such:
The lagging strand is looped
This allows the attached DNA polymerase to synthesize the Okazaki fragments in the normal 5’ to 3’ direction
Upon completion of an Okazaki fragment, the enzyme releases the lagging template strand
Another loop is then formed
This processed is repeated over and over again
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42. 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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43. 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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44. 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
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45. 11-45 Figure 11.14 A schematic drawing of proofreading
Site where DNA backbone is cut
A schematic drawing of proofreading
Figure 11.14
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46. 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 11.16
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47. Twice as many DnaA boxes
Insufficient amount of DnaA proteins
Some degraded; some stuck to cell membrane
The amount of DNA protein provides a way to regulate DNA replication
Figure 11.15
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48. 11-48 Figure 11.16 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 11.16
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49. 11-49 Figure 11.16 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 11.16
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50. Experiment 11B: DNA Replication Can Be Studied In Vitro
The in vitro study of DNA replication was pioneered by Arthur Kornberg in the 1950s
He received a Nobel Prize for his efforts in 1959
Kornberg hypothesized that deoxynucleoside triphosphates are the precursors of DNA synthesis
He also knew that these nucleotides are soluble in an acidic solution while long DNA strands are not
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51. Experiment 11B: DNA Replication Can Be Studied In Vitro
In this experiment, Kornberg mixed the following
An extract of proteins from E. coli
Template DNA
Radiolabeled nucleotides
These were incubated for sufficient time to allow the synthesis of new DNA strands
Addition of acid will precipitate these DNA strands
Centrifugation will separate them from the radioactive nucleotides
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52. Testing the Hypothesis
DNA synthesis can occur in vitro if all the necessary components are present
Testing the Hypothesis
Refer to Figure 11.17
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53. Figure 11.17
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54. Figure 11.17
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55. The Data 11-55 Conditions Amount of radiolabeled DNA* Complete system
3,300
Template DNA omitted
0.0
*Calculated in picomoles of 32P-labeled DNA
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56. Interpreting the Data 11-56 Conditions Amount of radiolabeled DNA*
E. coli proteins + nonlabeled template DNA + radiolabled nucleotides
= Radiolabeled product
Conditions
Amount of radiolabeled DNA*
Complete system
3,300
Template DNA omitted
0.0
*Calculated in picomoles of 32P-labeled DNA
Expected result because the template is necessary for replication
Taken together, these results indicate that this technique can be used to measure the synthesis of DNA in vitro
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57. Isolation of Mutants
The isolation of mutants has been crucial in elucidating DNA replication
For example, mutants played key roles in the discovery of
DNA pol III
Various other enzymes involved in DNA synthesis
DNA replication is vital for cell division
Thus, most mutations that block DNA synthesis are lethal
For this reason, researchers must screen for conditional mutants
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58. Isolation of Mutants
A type of conditional mutant is a temperature-sensitive (ts) mutant
In the case of a vital gene
A ts mutant can survive at the permissive temperature
But it will fail to grow at the nonpermissive one
Figure shows a general strategy for the isolation of ts mutants
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59. To increase the likelihood of mutations
Figure 11.18
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60. Isolation of Mutants
E. coli has many vital genes that are not involved in DNA replication
So only a subset of ts mutants would carry mutations affecting the replication process
Therefore, researchers in the 1960s had to screen thousands of ts mutants to get to those involved in DNA replication
This is sometimes called a “brute force” genetic screen
Table 11.3 summarizes some of the genes that were identified using this strategy
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61. 11-61
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62. Isolation of Mutants
The isolation of dna mutants was important in several ways
1. It allowed for the identification of the proteins that were defective in the mutant
2. It allowed for the mapping of these mutations along the E. coli chromosome
3. It provided an important starting point for the subsequent cloning and sequencing of these genes
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63. 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 1.1 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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64. 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 11.19
DNA replication proceeds bidirectionally from many origins of replication
Refer to Figure 11.20
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65. Bidrectional DNA synthesis Replication forks will merge
Figure 11.20
Part (b) shows a micrograph of a replicating DNA chromosome
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66. 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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67. Multiple Origins of Replication
Origin recognition complex (ORC)
A six-subunit complex that acts as the initiator of eukaryotic DNA replication
It appears to be found in all eukaryotes
Requires ATP to bind ARS elements
Single-stranded DNA stimulates ORC to hydrolyze ATP
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68. 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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69. 11-69
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70. 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 11.21
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71. Figure 11.21
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72. 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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73. Nucleosomes and DNA Replication
Replication doubles the amount of DNA
Therefore the cell must synthesize more histones to accommodate this increase
Synthesis of histones occurs during the S phase
Histones assemble into octamer structures
They associate with the newly made DNA very near the replication fork
Thus following DNA replication, each daughter strand has a mixture of “old” and “new” histones
Refer to Figure 11.22
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74. Newly made histone proteins
Figure 11.22
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75. 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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76. Telomeric sequences consist of
Moderately repetitive tandem arrays
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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77. 11-77

78. 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
Figure 11.24
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79. 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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80. The binding-polymerization-translocation cycle can occurs many times
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 complementary
strand is made by primase, DNA polymerase and ligase
Figure 11.25
RNA primer
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