1. How is DNA Replicated?

2. Once the structure of DNA was understood, it was possible to investigate how the molecule replicated itself during cell division.
In their paper, Watson and Crick allude to this possibility that the molecule suggests certain mechanism of replication
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3. DNA can be synthesized in a test tube if the following substances are present:
Deoxyribonucleoside triphophates, dNTPs of
dATP, dCTP, dGTP, dTTP
DNA template to direct formation of new molecules
DNA polymerase an enzyme to catalyze the polymerization reaction
Salts and pH buffer to create proper chemical environment.
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4. If DNA could be synthesized in a test tube, this indicates that DNA contains all the information needed for its own replication
So how does it do this?
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5. Three modes of DNA replication appear possible:
Conservative
Original helix is conserved and two newly synthesized strands come together
Semiconservative
Each replicated DNA molecule consists of one "old" strand and one new strand
Dispersive
Parental strands are dispersed into two new double helices
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6. Figure 11-2 Results of one round of replication of DNA for each of the three possible modes by which replication could be accomplished.
Figure 11.2
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7. An elegant experiment demonstrated that DNA replication is semiconservative
Meselson and Stahl (1958), using 15N-labeled E. coli grown in medium containing 14N, demonstrated that
DNA replication is semiconservative in prokaryotes
each new DNA molecule consists of one old strand and one newly synthesized strand
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8. The key is the use of “heavy” isotope of nitrogen – 15N
The key is the use of “heavy” isotope of nitrogen – 15N. Molecules with this heavy form of N are denser than those containing the more common isotope 14N
DNA extracts from the E. coli cultures could be centrifuged to separate the heavy strands from the light strands.
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9. Figure 11-3 The Meselson–Stahl experiment.
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10. Figure 11-4 The expected results of two generations of semiconservative replication in the Meselson–Stahl experiment.
Figure 11.4
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11. The results obtained can only be explained by the semiconservative model.
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12. Three Attributes of DNA Replication Shared by All Organisms
Each strand of the parental DNA molecule remains intact during replication
Each parental strand serves as a template for formation of an antiparallel, complementary daughter strand
Completion of replication results in the formation of two identical daughter duplexes composed of one parental and one daughter strand 12

13. 2 General Steps to DNA Replication
DNA replication in the cell requires many different enzymes and proteins.
DNA double helix is unwound to separate two template strands
New nucleotides form complementary base pairs with template DNA forming phosphodiester bonds
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14. Figure 11-7 The chemical reaction catalyzed by DNA polymerase I
Figure 11-7 The chemical reaction catalyzed by DNA polymerase I. During each step, a single nucleotide is added to the growing complement of the DNA template, using a nucleoside triphosphate as the substrate. The release of inorganic pyrophosphate drives the reaction energetically.
Figure 11.7
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15. As the nucleotide is added, the two terminal phosphates are cleaved off, providing a newly exposed 3'-OH group that can participate in the addition of another nucleotide as DNA synthesis proceeds
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16. Figure 11-8 Demonstration of to synthesis of DNA.
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18. DNA replication starts when a large protein complex (pre-replication complex) binds to a region called origin of replication (ori).
In E. coli, DNA is unwound and replication proceeds in both directions, forming two replication forks.
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19. Figure 11-6 Bidirectional replication of the E. coli chromosome
Figure 11-6 Bidirectional replication of the E. coli chromosome. The thin black arrows identify the advancing replication forks. The micrograph is of a bacterial chromosome in the process of replication, comparable to the figure next to it.
Figure 11.6
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20. In eukaryotes, there are more than one origin of replication along linear chromosomes
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21. Many Complex Issues Must Be Resolved during DNA Replication
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22. There are seven key issues that must be resolved during DNA replication:
Unwinding of the helix
Reducing increased coiling generated during unwinding
Synthesis of a primer for initiation
Discontinuous synthesis of the second strand
Removal of the RNA primers
Joining of the gap-filling DNA to the adjacent strand
Proofreading
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23. DnaA binds to the origin of replication and is responsible for the initial steps in unwinding the helix
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24. Subsequent binding of DnaB and DnaC further opens and destabilizes the helix
Proteins such as these, which require the energy normally supplied by the hydrolysis of ATP to break hydrogen bonds and denature the double helix, are called helicases
Single-stranded binding proteins (SSBPs) stabilize the open conformation
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25. Unwinding produces supercoiling that is relieved by DNA gyrase, a member of a larger group of enzymes referred to as DNA topoisomerases
Gyrase makes single- or double-stranded cuts to undo the twists and knots created during supercoiling, which are then resealed
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26. DNA polymerase I removes the primer and replaces it with DNA
To elongate a polynucleotide chain, DNA polymerase III requires a primer with a free 3'-hydroxyl group
Primase synthesizes an RNA primer that provides the free 3'-hydroxyl required by DNA polymerase III
DNA polymerase I removes the primer and replaces it with DNA
Priming is a universal phenomenon during initiation of DNA synthesis
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27. Figure 11-10 The initiation of DNA synthesis
Figure The initiation of DNA synthesis. A complementary RNA primer is first synthesized, to which DNA is added. All synthesis is in the to direction. Eventually, the RNA primer is replaced with DNA under the direction of DNA polymerase I.
Figure 11.10
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28. The opposite lagging strand undergoes discontinuous DNA synthesis
As the replication fork moves, only one strand can serve as a template for continuous DNA synthesis—the leading strand
The opposite lagging strand undergoes discontinuous DNA synthesis
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29. Figure Opposite polarity of DNA synthesis along the two strands, necessary because the two strands of DNA run antiparallel to one another and DNA polymerase III synthesizes only in one direction ( to ). On the lagging strand, synthesis must be discontinuous, resulting in the production of Okazaki fragments. On the leading strand, synthesis is continuous. RNA primers are used to initiate synthesis on both strands.
Figure 11.11
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30. The lagging strand is synthesized as Okazaki fragments, each with an RNA primer
DNA polymerase I removes the primers on the lagging strand, and the fragments are joined by DNA ligase
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31. A model of DNA Replication
DNA synthesis at a single replication fork involves
DNA polymerase III
single-stranded binding proteins
DNA gyrase
DNA helicase
RNA primers
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32. Figure 11-13 Summary of DNA synthesis at a single replication fork
Figure Summary of DNA synthesis at a single replication fork. Various enzymes and proteins essential to the process are shown.
Figure 11.13
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33. Checkpoint
In the figure shown, the leading strand grows continuously _______ at its _______ end.
backward; 5′
forward; 5′
backward; 3′
forward; 3′
Answer: d

34. Checkpoint Strand A Strand B5’ 3’
Strand B
In the figure shown, what best describes Strand B?
It is the lagging strand.
It is the leading strand.
It is the Okazaki strand.
There is not enough information given.
Answer: b

35. Eukaryotic DNA Replication Is Similar to Replication in Prokaryotes, but Is More Complex
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36. Eukaryotic DNA replication shares many features with replication in bacteria
Double-stranded DNA unwound at replication origins
Replication forks are formed
Bidirectional snythesis creates leading and lagging strands
Eukaryotic polymerases require four deoxyribonucleoside triphosphates, a template, and a primer
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37. This makes DNA replication more complex in eukaryotes than in bacteria
However, eukaryotic DNA replication is more complex due to several features of eukaryotic DNA:
There is more DNA than prokaryotic cells
The chromosomes are linear
The DNA is complexed with proteins
This makes DNA replication more complex in eukaryotes than in bacteria
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38. Three DNA polymerases are involved in replication of nuclear DNA
One involves mitochondrial DNA replication
Others are involved in repair processes
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39. Visit Blackboard content and review the media file “ DNA Replication”
Stop and view:
Visit Blackboard content and review the media file “ DNA Replication”
This is a very helpful animation of the steps of replication.
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40. The Ends of Linear Chromosome Are Problematic during Replication
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41. Telomeres at the ends of linear chromosomes consist of long stretches of short repeating sequences and preserve the integrity and stability of chromosomes
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42. Lagging strand synthesis at the end of the chromosome is a problem because once the RNA primer is removed, there is no free 3'-hydroxyl group from which to elongate
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43. Telomerase directs synthesis of the telomere repeat sequence to fill the gap
This enzyme is a ribonucleoprotein with an RNA that serves as the template for the synthesis of its DNA complement
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44. In most eukaryotic somatic cells, telomerase is not active
With each successive cell division, telomeres shorten and erode, causing further cell division to stop
Malignant cells maintain telomerase activity and are immortalized
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45. Telomere length is important for chromosome stability, cell longevity, and reproductive success
Telomerase is active in germ-line cells and some stem cells in eukaryotes
Differentiated somatic cells and cells in culture have virtually no telomerase activity; such cells have limited life spans (30 to 50 cell divisions)
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46. Telomerase is normally turned off in somatic cells
Reactivation of telomerase can lead to aging cells that continue to proliferate, a feature of many types of cancer
TERT reactivation is one of the most common mutations in cancers of all types
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47. Errors in DNA repaired
DNA must be faithfully maintained, yet replication is not perfectly accurate and DNA is subject to damage by chemicals and other environmental agents.
Cells have three mechanisms for DNA repair:
Proofreading corrects errors in replication as DNA polymerase makes them
Mismatch repair scans DNA immediately after replication and corrects any base pairing
Excision repair removes abnormal bases that have formed because of chemical damage and replaces them with functional ones.
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48. Homework Assignment
Page 289 in your textbook: Telomeres: Key to Immortality. Under “Your Turn” complete questions #4 and submit to your Box Folder
See mastering genetics for homework problems. Mastering assignment has been set as tutorial homework. There is no deductions for wrong answers.
Both are due by Monday, March 3, at 9:00 PM
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