1. It is the process by which the genetic material is copied
DNA replication
It 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
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2. 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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3. A pairs with T and G with C to form new strand
Identical base sequences

4. 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. 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 two replication forks eventually meet at the opposite side of the bacterial chromosome
This ends replication
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8. 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
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10. DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences
This binding stimulates the cooperative binding of additional ATP-bound DnaA proteins to form a large complex
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

11. Travels along the DNA in the 5’ to 3’ direction
Uses energy from ATP
Bidirectional replication

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14. 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
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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15. 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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16. 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
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17. 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

18. Leading strand Lagging strand
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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19. 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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21. 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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22. Innermost phosphate

23. 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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24. 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
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25. 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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26. Helicase and Primase are linked together in a “primosome”
Two DNA polymerase III holoenzymes and the primosome are linked together to form the Replisome

27. 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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28. 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 process is repeated over and over again
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29. 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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30. 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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31. 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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32. A schematic drawing of proofreading
Site where DNA backbone is cut
A schematic drawing of proofreading

33. 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
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
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34. 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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35. 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
DNA replication proceeds bidirectionally from many origins of replication
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36. 100 μm Chromosome Sister chromatids Origin Origin Origin Centromere
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100 μm
© This article was published in Journal of Molecular Biology. Mar 14;32(2). Huberman JA. Riggs AD. “Links On the mechanism of DNA replication in mammalian chromosomes." Copyright Elsevier, Reprinted with permission.
Chromosome
Sister chromatids
Origin
Origin
Origin
Centromere
Origin
Origin
Before S phase
During S phase
End of S phase

37. 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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38. Eukaryotes Contain Several Different DNA Polymerases
Mammalian cells contain well over a dozen different DNA polymerases
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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39. 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 e or d for the processive elongation of the leading and lagging strands, respectively
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
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40. 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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41. Flap Endonuclease Removes RNA Primers
This is function of Pol I in bacteria
Polymerase d runs into primer of adjacent Okazaki fragment
Pushes portion of primer into short flap
Flap endonuclease removes the primer
Long flaps are removed by Dna2 nuclease
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42. 5′ 3′ 3′ 5′
DNA polymerase δ elongates the
left Okazaki fragment and causes
a short flap to occur on the right
Okazaki fragment.
Flap 5′ 3′ 3′ 5′
Flap endonuclease
removes the flap. 5′ 3′ 3′ 5′
DNA polymerase δ
continues to elongate and
causes a second flap. 5′ 3′ 3′ 5′
Flap endonuclease
removes the flap. 5′ 3′ 3′ 5′
Process continues until the
entire RNA primer is removed.
DNA ligase seals the two
fragments together. 5′ 3′ 3′ 5′
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43. 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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44. Telomeric sequences consist of
Moderately repetitive tandem arrays
3’ overhang that is nucleotides long
Telomeric sequences typically consist of
Several guanine nucleotides
Often many thymine nucleotides
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46. 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!

47. 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
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48. Telomerase reverse transcriptase (TERT) activity
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
RNA primer