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Table of contents

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1. Introduction to Genetics51m
2. Mendel's Laws of Inheritance3h 37m
3. Extensions to Mendelian Inheritance2h 41m
4. Genetic Mapping and Linkage2h 28m
5. Genetics of Bacteria and Viruses1h 21m
6. Chromosomal Variation1h 48m
7. DNA and Chromosome Structure56m
8. DNA Replication1h 10m
9. Mitosis and Meiosis1h 34m
10. Transcription1h 0m
11. Translation58m
12. Gene Regulation in Prokaryotes1h 19m
13. Gene Regulation in Eukaryotes44m
14. Genetic Control of Development44m
15. Genomes and Genomics1h 50m
16. Transposable Elements47m
17. Mutation, Repair, and Recombination1h 6m
18. Molecular Genetic Tools19m
- Genetic Cloning
  9m
- Methods for Analyzing DNA
  9m
19. Cancer Genetics29m
- Overview of Cancer
  21m
- Cancer Mutations
  7m
20. Quantitative Genetics1h 26m
21. Population Genetics50m
- Hardy Weinberg
  19m
- Allelic Frequency Changes
  31m
22. Evolutionary Genetics29m

8. DNA Replication

Overview of DNA Replication

8. DNA Replication

Overview of DNA Replication

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Proofreading

Kylia Goodner

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Okay. So now let's talk about DNA proofreading. If you know anything about DNA replication, you know it is replicated with extremely high fidelity. It very rarely makes an error. And the reason is that there's one error every 1010 nucleotides, and this happens very quickly. Right? Nearly a 1000 nucleotides are replicated per strand, so that means 2,000 nucleotides per second. Not only is it very accurate, but it's also occurring very fast. It has this accuracy at very high speeds. And the reason it's so accurate is because DNA polymerase has proofreading abilities. So it's not always perfect. Right? Sometimes, that's called a DNA mismatch. If a DNA mismatch is made, the polymerase actually pauses, goes back, cuts out that wrong base, and replaces it. And this type of activity is called exonuclease activity, and it's very important you know that it occurs 3′ to 5′ prime. Know this because this is opposite. Right? So synthesis on the new strand occurs 5′ to 3′ prime, the proofreading occurs 3′ to 5′ prime. It's important to understand these differences and the directional differences. And so, the exonuclease just means that it can cut out a mismatched nucleotide. So here we have an example of what this looks like. We have DNA polymerase. It's going forth. It's just replicating itself and it's so happy. And here it has made an error. Right? C doesn't go with T. A goes with T and C goes with G. So this is a big error. What it does is it actually pauses, goes back, cuts that nucleotide out, but it cuts that C out that's been paired wrong, using its 3′ to 5′ exonuclease activity, and then it replaces it and just keeps going. And so this proofreading ability is super important in giving DNA polymerase the high fidelity that we need to survive. If it didn't have this, we would have a much higher error rate. We would have a lot more mutations, a lot more health problems, essentially, and we may not even be able to survive as humans without this proofreading ability. So with that, let's now move on.

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Related Practice

Textbook Question

What observations reveal that a 'telomere problem' exists during eukaryotic DNA replication, and how did we learn of the solution to this problem?

Textbook Question

In this chapter, we focused on how DNA is replicated and synthesized. We also discussed recombination at the DNA level. Along the way, we encountered many opportunities to consider how this information was acquired. On the basis of these discussions, what answers would you propose to the following fundamental questions? How do we know that DNA synthesis is discontinuous on one of the two template strands?

Textbook Question

In this chapter, we focused on how DNA is replicated and synthesized. We also discussed recombination at the DNA level. Along the way, we encountered many opportunities to consider how this information was acquired. On the basis of these discussions, what answers would you propose to the following fundamental questions? How do we know that in vivo DNA synthesis occurs in the 5' to 3' direction?

Textbook Question

In this chapter, we focused on how DNA is replicated and synthesized. We also discussed recombination at the DNA level. Along the way, we encountered many opportunities to consider how this information was acquired. On the basis of these discussions, what answers would you propose to the following fundamental questions? How was it demonstrated that DNA synthesis occurs under the direction of DNA polymerase III and not polymerase I?

Textbook Question

Write a short essay that distinguishes between the terms replication and synthesis, as applied to DNA. Which of the two is most closely allied with the field of biochemistry?

Textbook Question

Compare conservative, semiconservative, and dispersive modes of DNA replication.

Textbook Question

Describe the role of ¹⁵N in the Meselson–Stahl experiment.

Textbook Question

Predict the results of the experiment by Taylor, Woods, and Hughes if replication were (a) conservative and (b) dispersive.

Textbook Question

What are the requirements for in vitro synthesis of DNA under the direction of DNA polymerase I?

Textbook Question

In Kornberg's initial experiments, it was rumored that he grew E. coli in Anheuser-Busch beer vats. (Kornberg was working at Washington University in St. Louis.) Why do you think this might have been helpful to the experiment?

Textbook Question

How did Kornberg assess the fidelity of DNA polymerase I in copying a DNA template?

Textbook Question

Which characteristics of DNA polymerase I raised doubts that its in vivo function is the synthesis of DNA leading to complete replication?

Textbook Question

DNA polymerase III is the main DNA-synthesizing enzyme in bacteria. Describe how it carries out its role of elongating a strand of DNA.

Textbook Question

Kornberg showed that nucleotides are added to the 3' end of each growing DNA strand. In what way does an exposed 3'-OH group participate in strand elongation?

Textbook Question

What was the significance of the polA1 mutation?

Textbook Question

Explain how RNA participates in DNA replication.

Textbook Question

Summarize and compare the properties of DNA polymerase I, II, and III.

Textbook Question

A sample of double-stranded DNA is found to contain 20% cytosine. Determine the percentage of the three other DNA nucleotides in the sample.

Textbook Question

List and describe the function of the ten subunits constituting DNA polymerase III. Distinguish between the holoenzyme and the core enzyme.

Textbook Question

Bacterial DNA polymerase I and DNA polymerase III perform different functions during DNA replication.

If a strain of E. coli acquired a mutation that inactivated DNA polymerase III function, would the cell be able to replicate its DNA? Why or why not?

Textbook Question

Bacterial DNA polymerase I and DNA polymerase III perform different functions during DNA replication.

Identify the principal functions of each molecule.

Textbook Question

Bacterial DNA polymerase I and DNA polymerase III perform different functions during DNA replication.

If mutation inactivated DNA polymerase I in a strain of E. coli, would the cell be able to replicate its DNA? If so, what kind of abnormalities would you expect to find in the cell?

Textbook Question

Distinguish between (a) unidirectional and bidirectional synthesis, and (b) continuous and discontinuous synthesis of DNA.

Textbook Question

Diagram a replication fork in bacterial DNA and label the following structures or molecules.

a. DNA pol III
b. helicase
c. RNA primer
d. origin of replication
e. leading strand (label its polarity)
f. DNA pol I
g. topoisomerase
h. SSB protein
i. lagging strand (label its polarity)
j. primase
k. Okazaki fragment

Textbook Question

List the proteins that unwind DNA during in vivo DNA synthesis. How do they function?

Textbook Question

Define and indicate the significance of (a) Okazaki fragments, (b) DNA ligase, and (c) primer RNA during DNA replication.

Textbook Question

Outline the current model for DNA synthesis.

Textbook Question

Why is DNA synthesis expected to be more complex in eukaryotes than in bacteria? How is DNA synthesis similar in the two types of organisms?

Textbook Question

Suppose that E. coli synthesizes DNA at a rate of 100,000 nucleotides per minute and takes 40 minutes to replicate its chromosome. (a) How many base pairs are present in the entire E. coli chromosome? (b) What is the physical length of the chromosome in its helical configuration—that is, what is the circumference of the chromosome if it were opened into a circle?

Textbook Question

Several temperature-sensitive mutant strains of E. coli display the following characteristics. Predict what enzyme or function is being affected by each mutation. Supercoiled strands remain after replication, which is never completed.

Textbook Question

Several temperature-sensitive mutant strains of E. coli display the following characteristics. Predict what enzyme or function is being affected by each mutation. Synthesis is very slow.

Textbook Question

Several temperature-sensitive mutant strains of E. coli display the following characteristics. Predict what enzyme or function is being affected by each mutation. No initiation occurs.

Textbook Question

Several temperature-sensitive mutant strains of E. coli display the following characteristics. Predict what enzyme or function is being affected by each mutation. Okazaki fragments accumulate, and DNA synthesis is never completed.

Textbook Question

Several temperature-sensitive mutant strains of E. coli display the following characteristics. Predict what enzyme or function is being affected by each mutation. Newly synthesized DNA contains many mismatched base pairs.

Textbook Question

DNA supercoiling, which occurs when coiling tension is generated ahead of the replication fork, is relieved by DNA gyrase. Supercoiling may also be involved in transcription regulation. Researchers discovered that enhancers operating over a long distance (2500 bp) are dependent on DNA supercoiling, while enhancers operating over shorter distances (110 bp) are not so dependent [Liu et al. (2001). Proc. Natl. Acad. Sci. USA 98:14,883–14,888]. Using a diagram, suggest a way in which supercoiling may positively influence enhancer activity over long distances.

Textbook Question

Raymond Rodriguez and colleagues demonstrated conclusively that DNA replication in E. coli is bidirectional. Explain why locating the origin of replication on one side of the circular chromosomes and the terminus of replication on the opposite side of the chromosome supported this conclusion.

Textbook Question

While many commonly used antibiotics interfere with protein synthesis or cell wall formation, clorobiocin, one of several antibiotics in the aminocoumarin class, inhibits the activity of bacterial DNA gyrase. Similar drugs have been tested as treatments for human cancer. How might such drugs be effective against bacteria as well as cancer?

Textbook Question

Joel Huberman and Arthur Riggs used pulse–chase labeling to examine the replication of DNA in mammalian cells. Briefly describe the Huberman–Riggs experiment, and identify how the results exclude a unidirectional model of DNA replication.

Textbook Question

Why do the genomes of eukaryotes, such as Drosophila, need to have multiple origins of replication, whereas bacterial genomes, such as that of E. coli, have only a single origin?

Textbook Question

Bloom syndrome (OMIM 210900) is an autosomal recessive disorder caused by mutation of a DNA helicase. Among the principal symptoms of the disease are chromosome instability and a propensity to develop cancer. Explain these symptoms on the basis of the helicase mutation.

Textbook Question

How does rolling circle replication (see Section 6.2) differ from bidirectional replication?

Textbook Question

The genome of D. melanogaster consists of approximately 1.7x10⁸ base pairs. DNA synthesis occurs at a rate of 30 base pairs per second. In the early embryo, the entire genome is replicated in five minutes. How many bidirectional origins of synthesis are required to accomplish this feat?

Textbook Question

DNA polymerases in all organisms add only 5' nucleotides to the 3' end of a growing DNA strand, never to the 5' end. One possible reason for this is the fact that most DNA polymerases have a proofreading function that would not be energetically possible if DNA synthesis occurred in the 3' to 5' direction. Consider the information in your sketch and speculate as to why proofreading would be problematic.

Textbook Question

DNA polymerases in all organisms add only 5' nucleotides to the 3' end of a growing DNA strand, never to the 5' end. One possible reason for this is the fact that most DNA polymerases have a proofreading function that would not be energetically possible if DNA synthesis occurred in the 3' to 5' direction. Sketch the reaction that DNA polymerase would have to catalyze if DNA synthesis occurred in the 3' to 5' direction.

Textbook Question

Assume that the sequence of bases shown below is present on one nucleotide chain of a DNA duplex and that the chain has opened up at a replication fork. Synthesis of an RNA primer occurs on this template starting at the base that is underlined. In the intact RNA primer, which nucleotide has a free 3'-OH terminus? 3'.......GGCTACCTGGATTCA....5'

Textbook Question

Assume that the sequence of bases shown below is present on one nucleotide chain of a DNA duplex and that the chain has opened up at a replication fork. Synthesis of an RNA primer occurs on this template starting at the base that is underlined. If the RNA primer consists of eight nucleotides, what is its base sequence?

Textbook Question

Reiji and Tuneko Okazaki conducted a now classic experiment in 1968 in which they discovered a population of short fragments synthesized during DNA replication. They introduced a short pulse of ³H-thymidine into a culture of E. coli and extracted DNA from the cells at various intervals. In analyzing the DNA after centrifugation in denaturing gradients, they noticed that as the interval between the time of ³H-thymidine introduction and the time of centrifugation increased, the proportion of short strands decreased and more labeled DNA was found in larger strands. What would account for this observation?

Textbook Question

DNA replication in early Drosophila embryos occurs about every 5 minutes. The Drosophila genome contains approximately 1.8×10⁸ base pairs. Eukaryotic DNA polymerases synthesize DNA at a rate of approximately 40 nucleotides per second. Approximately how many origins of replication are required for this rate of replication?

Textbook Question

What would be the effects on DNA replication if mutation of DNA pol III caused it to lose each of the following activities?

3′ to 5′ exonuclease activity

Textbook Question

What would be the effects on DNA replication if mutation of DNA pol III caused it to lose each of the following activities?

5' to 3' polymerase activity

Textbook Question

Suppose that future exploration of polar ice on Mars identifies a living microbe and that analysis indicates the organism carries double-stranded DNA as its genetic material. Suppose further that DNA replication analysis is performed by first growing the microbe in a growth medium containing the heavy isotope of nitrogen (¹⁴N) that the organism is then transferred to a growth medium containing the light isotope of nitrogen (¹⁴N) and that the nitrogen composition of the DNA is examined by CsCl ultracentrifugation and densitometry after the first, second, and third replication cycles in the ¹⁴N-containing medium. The results of the experiment are illustrated here for each cycle. The control shows the positioning of the three possible DNA densities. Based on the results shown, what can you conclude about the mechanism of DNA replication in this organism? (Hint: See the description of the Meselson and Stahl experiment on Section 6.1)

Textbook Question

The following diagram shows the parental strands of a DNA molecule undergoing replication. Draw the daughter strands present in the replication bubble, indicating a. The polarity of daughter strands b. The leading and lagging strands c. Okazaki fragments d. The locations of RNA primers

Multiple Choice

Which of the following proteins is responsible for unwinding the double stranded DNA?

Multiple Choice

DNA replication synthesizes DNA in which direction?

Multiple Choice

What would happen to DNA replication in DNA polymerase lost its 3' to 5' exonuclease activity?

Multiple Choice

Which of the following proteins is responsible for synthesizing RNA primers?

Multiple Choice

The short DNA fragments created during lagging strand replication are called what?

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