Deck 2: DNA Replication

Question 1

Describe the Meselson-Stahl experiment, and explain how it showed that DNA replication is semiconservative.

Answer 1

In 1958, Meselson and Stahl set out to determine the model of deoxyribonucleic acid (DNA) replication. First, they grew E. coli in a medium where the only nitrogen available was ammonium chloride in which the normal 14 N isotope is replaced with the heavier 15 N isotope: 15 NH 4 Cl. The heavier isotope is just slightly heavier than the normal one.
By growing the bacteria in such a medium, all the bacteria's nitrogen containing compounds including DNA would contain 15 N instead of 14 N. In the next step, the bacteria were transferred to a medium in which the nitrogen containing compounds contained only the 14 N form. The bacteria were then allowed to reproduce for a variable amount of generations.
Thus, Meselson and Stahl, after a certain number of generations determine how much 14 N versus 15 N were present in the offspring in order to determine the model of DNA replication. Samples of E. coli were taken after varying amounts of generations and the DNA was extracted and its density analyzed.
The experimenters used density gradient centrifugation, using cesium chloride (CsCl). In this method, a sample is centrifuged at high speed with CsCl resulting in gradient of salt in which the least dense solution is at the top of the tube while the more dense solution is at the bottom. The sample, usually composed of DNA will form a band where its buoyant density matches that of CsCl.
There will be a band for 14 N-labeled DNA and another band closer to the top of the solution for 15 N-labeled DNA.
When the experimenters studied the density of the DNA after one generation, they found that only a single band formed intermediate to purely 14 N-labeled DNA and to purely 15 N-labeled DNA. Thus, it was concluded that the first generation DNA was filled with half of both types of DNA which confirmed that DNA replicated in a semiconservative fashion. The first generation retained half its DNA from a parental strand.

Question 2

In the Meselson-Stahl experiment, 15 N-labeled cells were shifted to a 14 N medium at what we can designate as generation 0.
a. For the semiconservative model of replication, what proportion of 15 N- 15 N, 15 N- 14 N, and 14 N- 14 N DNA would you expect to find after one, two, three, four, six, and eight replication cycles?
b. Answer (a) in terms of the conservative model of DNA replication.

Answer 2

In an experiment, 15 N-labelled bacterial cells are shifted to a 14 N medium at generation 0.
a) The parent generation will have purely 15 N- 15 N deoxyribonucleic acid (DNA). When the DNA replicated to produce offspring, the two strands will split apart and create a complementary strand to each of them completely composed of 14 N-DNA. Thus, the first generation will have 100% 15 N- 14 N DNA.
When the first generation replicates, the strands will split apart and one parent strand will be 14 N-DNA and the other parent strand will be 15 N-DNA. Once the two parent strands gain their complement strands, the complement strand will be composed completely of 14 N-DNA. So, the second generation will have 50% 15 N- 14 N DNA and 50% 14 N- 14 N DNA.
With each successive generation the amount of DNA containing a mixture of nitrogen isotopes will be halved, and the remaining DNA will be composed of only the normal isotope.
In the third generation, the proportion of 15 N- 14 N DNA will be 25% and the proportion of 14 N- 14 N DNA will be 75%.
In the fourth generation, the proportion of 15 N- 14 N DNA will be 12.5% and the proportion of 14 N- 14 N DNA will be 87.5%.
In the sixth generation, the proportion of 15 N- 14 N DNA will be 3.125% and the proportion of 14 N- 14 N DNA will be 96.875%.
In the eighth generation, the proportion of 15 N- 14 N DNA will be.78125% and the proportion of 14 N- 14 N DNA will be 99.21875%.
b) In the conservative model of DNA replication, the parent retains its strands and passes on new strands to offspring. So, the parent generation will have purely 15 N- 15 N deoxyribonucleic acid (DNA). When a member of the parent generation replicates, one daughter cell will retain both parent strands, and one daughter cell will have new daughter strands composed of 14 N- 14 N DNA. Therefore, the first generation will consist of 50% 15 N- 15 N DNA and 50% 14 N- 14 N DNA.
With each successive generation, the heavier DNA will be passed to half the daughter cells. Thus, the proportion of the heavier 15 N- 15 N DNA will be halved each time. In the second generation, the proportion of 15 N- 15 N DNA will be 25% and the proportion of 14 N- 14 N DNA will be 75%.
In the third generation, the proportion of 15 N- 15 N DNA will be 12.5% and the proportion of 14 N- 14 N DNA will be 87.5%.
In the fourth generation, the proportion of 15 N- 15 N DNA will be 6.25% and the proportion of 14 N- 14 N DNA will be 93.75%.
In the sixth generation, the proportion of 15 N- 15 N DNA will be 1.5625% and the proportion of 14 N- 14 N DNA will be 98.4375%.
In the eighth generation, the proportion of 15 N- 15 N DNA will be.39% and the proportion of 14 N- 14 N DNA will be 99.61%.

Question 3

A spaceship lands on Earth, bringing with it a sample of extraterrestrial bacteria. You are assigned the task of determining the mechanism of DNA replication in this organism.
You grow the bacteria in an unlabeled medium for several generations and then grow it in the presence of for exactly one generation. You extract the DNA and subject it to CsCl centrifugation. The banding pattern you find is as follows:
It appears to you that this pattern is evidence that DNA replicates in the semiconservative manner, but you are wrong. Why? What other experiment could you perform (using the same sample and technique of CsCl centrifugation) that would further distinguish between semiconservative and dispersive modes of replication?

Answer 3

Even if the deoxyribonucleic acid (DNA) has a banding pattern intermediate to purely heavy 15 N- 15 N DNA and purely light 14 N- 14 N DNA, it is not an indication that the DNA is being replicated in a semiconservative manner, rather it is an indication that the first generation of offspring had DNA that is composed with equal proportions of 14 N and 15 N. It is possible that the DNA is passed on to offspring via the dispersive method, in which the double helix is cleaved in to segments that are used as templates for new strands.
In order to distinguish between the dispersive model and the semiconservative model, the CsCl centrifugation should be used on DNA samples for other generations after generation 0. If the DNA replicates under the dispersive model, then with each successive replication the 15 N- 15 N DNA would continue to be dispersed among 14 N- 14 N DNA. So, with each successive generation, there will be an intermediate band that grows lighter and lighter.
In contrast, in the semiconservative model of replication an intermediate band only appears from the first generation of offspring.

Question 4

The elegant Meselson-Stahl experiment was among the first experiments to contribute to what is now a highly detailed understanding of DNA replication. Consider this experiment again in light of current molecular models by answering the following questions:
a. Does the fact that DNA replication is semiconservative mean that it must be semidiscontinuous?
b. Does the fact that DNA replication is semidiscontinuous ensure that it is also semiconservative?
c. Do any properties of known DNA polymerases ensure that DNA is synthesized semiconservatively?

Question 5

List the components necessary to make DNA in vitro, using the enzyme system isolated by Kornberg.

Question 6

Each of the following templates is added to an in vitro DNA synthesis reaction using the enzyme system isolated by Kornberg with 5
ATG-3? as a primer.
In what ways besides their sequence will the synthesized molecules differ if a trace amount of each of the following nucleotides is added to the reaction?
a.
32 P-dATP (dATP where the phosphorus closest to the 5
carbon is radioactive)b. 32 P-dAMP (dAMP where the phosphorus is radioactive)c.
32 P-dATP (dATP where the phosphorus furthest from the 5
carbon is radioactive)

Question 7

How do we know that the Kornberg enzyme is not the main enzyme involved in DNA synthesis for chromosome duplication in the growth of E. coli ?

Question 8

Kornberg isolated DNA polymerase I from E. coli. What is the function of the enzyme in DNA replication?

Question 9

Suppose you have a DNA molecule with the base sequence TATCA, going from the 5? to the 3? end of one of the polynucleotide chains. The building blocks of the DNA are drawn as in the following figure:
Use this shorthand system to diagram the completed double-stranded DNA molecule, as proposed by Watson and Crick.

Question 10

Use the shorthand notation of Question to diagram how a strand with the sequence 3
GGTCTAA-5? would anneal to a primer having the sequence 5
AGA-3?. Then answer the following questions.
a. What chemical groups do you expect to find at the and ends of each DNA strand?
b. What nucleotides would be used to extend the primer if the annealed DNA molecules are added to an in vitro DNA synthesis reaction using the system established by Kornberg?
c. What is the source of the energy used to catalyze the formation of phosphodiester bonds in the synthesis reaction in part (b)?
d. On a distant planet, cellula3 life is found to have a novel DNA polymerase that synthesizes a complementary DNA strand from a primed, single-stranded template, but does so only in the 5?to 5? direction. What nucleotides would be added to the primer if the annealed DNAs were present in a cell with this polymerase?
e. Reflect on your answer to part (c). Do you think the novel DNA polymerase catalyzes the formation of phosphodiester bonds in the same way as Earth DNA polymerases? If not, how might it catalyze the formation of phosphodiester bonds?
f. It would be faster if DNA polymerases could synthesize DNA in both the 3
to-5? and 5
to-3? directions. Speculate on why no known Earth DNA polymerase can synthesize DNA in both directions even though this seems to be a desirable trait.
Question
Suppose you have a DNA molecule with the base sequence TATCA, going from the 5? to the 3? end of one of the polynucleotide chains. The building blocks of the DNA are drawn as in the following figure:
Use this shorthand system to diagram the completed double-stranded DNA molecule, as proposed by Watson and Crick.

Question 11

Listed below are three enzymatic properties of DNA polymerases.
1. All DNA polymerases replicate DNA only 5? tO₃?.
2. During DNA replication, DNA polymerases synthesize DNA from an RNA primer.
3. Only some DNA polymerases have 5
to-3? exonuclease activity.
Explain whether each of these properties constrains DNA replication to be
a. semiconservative.
b. semidiscontinuous.

Question 12

Base analogs are compounds that resemble the natural bases found in DNA and RNA but are not normally found in those macromolecules. Base analogs can replace their normal counterparts in DNA during in vitro DNA synthesis. Researchers studied four base analogs for their effects on in vitro DNA synthesis using E. coli DNA polymerase. The results were as follows, with the amounts of DNA synthesized expressed as percentages of the DNA synthesized from normal bases only:
Which bases are analogs of adenine? of thymine? of cytosine? of guanine?

Question 13

Concerning DNA replication:
a. Describe (draw) models of continuous, semidiscontinuous, and discontinuous DNA replication.
b. What was the contribution of Reiji and Tuneko Okazaki and colleagues with regard to these replication models?

Question 14

The following events, steps, or reactions occur during E. coli DNA replication. For each entry in column A, select its match(es) from column B. Each entry in A may have more than one match, and each entry in B can be used more than once.

Question 15

Distinguish between the actions of helicase and topoisomerase on double-stranded DNA and their roles during DNA replication.

Question 16

How long would it take E. coli to replicate its entire genome (4.2 x 10 6 bp) , assuming a replication rate of 1,000 nucleotides per second at each fork with no pauses?

Question 17

A diploid organism has 4.5 x 10 8 bp in its DNA. The DNA is replicated in 3 minutes. Assuming that all replication forks move at a rate of 10 4 bp per minute, how many replicons (replication units) are present in the organism's genome?

Question 18

Describe the molecular action of the enzyme DNA ligase. What properties would you expect an E. coli cell to have if it had a temperature-sensitive mutation in the gene for DNA ligase?

Question 19

Chromosome replication in E. coli commences from a constant point, called the origin of replication. It is known that DNA replication is bidirectional. Devise a biochemical experiment to prove that the E. coli chromosome replicates bidirectionally. (Hint: Assume that the amount of gene product is directly proportional to the number of genes.)

Question 20

Reiji Okazaki concluded that both DNA strands could not replicate continuously. What evidence led him to this conclusion?

Question 21

A space probe returns from Jupiter and brings with it a new microorganism for study. It has double-stranded DNA as its genetic material. However, studies of replication of the alien DNA reveal that, although the process is semiconservative, DNA synthesis is continuous on both the leading-strand and the lagging-strand templates. What conclusions can you draw from this result?

Question 22

A space probe returning from Europa, one of Jupiter's moons, carries back an organism having linear chromosomes composed of double-stranded DNA. Like Earth organisms, its DNA replication is semiconservative. However, it has just one DNA polymerase, and this polymerase initiates DNA replication only at one, centrally located site using a DNA-primed template strand.
a. What enzymatic properties must its DNA polymerase have?
b. How is DNA replication in this organism different from DNA replication in E. coli, which is also initiated at just one site?

Question 23

Some phages, such as ?, are packaged from concatamers.
a. What is a concatamer, and what type of DNA replication is responsible for producing a concatamer?
b. In what ways does this type of DNA replication differ from that used by E. coli?

Question 24

Although ? is replicated into a concatamer, linear unit-length molecules are packaged into phage heads.
a. What enzymatic activity is required to produce linear unit-length molecules, how does it produce molecules that contain a single complete ? genome, and what gene encodes the enzyme involved?
b. What types of ends are produced when this enzyme acts on DNA, and how are these ends important in the ? life cycle?

Question 25

M13 is an E. coli bacteriophage whose capsid holds a closed circular DNA molecule witH₂,221 T, 1,296 C, 1,315 G, and 1,575 A nucleotides. M13 lacks a gene for DNA polymerase and so must use bacterial DNA polymerases for replication. Unlike ? this phage does not form concatamers during replication and packaging.
a. Suppose the M13 chromosome were replicated in a manner similar to the way the E. coli chromosome is replicated, using semidiscontinuous replication from a double-stranded circular DNA template. How would the semidiscontinuous DNA replication mechanism discussed in the text need to be modified?
b. Suppose the M13 chromosome were replicated in a manner similar to the way the chromosome is replicated, using rolling circle replication. How would the rolling circle replication mechanism discussed in the text need to be modified?

Question 26

Compare and contrast eukaryotic and prokaryotic DNA polymerases.

Question 27

What mechanism do eukaryotic cells employ to keep their chromosomes from replicating more than once per cell cycle?

Question 28

A mutation occurs that results in the failure of licensing factors to be inactivated after they are released from prereplicative complexes. What molecular consequences do you predict for this mutation?

Question 29

Autoradiography is a technique that allows radioactive areas of chromosomes to be observed under the microscope. The slide is covered with a photographic emulsion, which is exposed by radioactive decay. In regions of exposure, the emulsion forms silver grains on being developed. The tiny silver grains can be seen on top of the (much larger) chromosomes. Devise a method to find out which regions in the human karyotype replicate during the last 30 minutes of the S phase. (Assume a cell cycle in which the cell spends 10 hours in G 1 , 9 hours in S, 4 hours in G 2 and 1 hour in M.)

Question 30

In typical human fibroblasts in culture, the G 1 , period of the cell cycle lasts about 10 hours, S lasts about 9 hours, G 2 takes 4 hours, and M takes 1 hour. Suppose you added radioactive ( 3 H) thymidine to the medium, left it there for 5 minutes, and then washed it out and replaced it with an ordinary medium.
a. What percentage of cells would you expect to become labeled by incorporating the 3 H-thymidine into their DNA?
b. How long would you have to wait after removing the medium before you would see labeled metaphase chromosomes?
c. Would one or both chromatids be labeled?
d. How long would you have to wait if you wanted to see metaphase chromosomes containing ( 3 H) in the regions of the chromosomes that replicated at the beginning of the S period?

Question 31

Suppose you performed the experiment in Question.30, but left the radioactive medium on the cells for 16 hours instead of 5 minutes. How would your answers change?

Question 32

How is chromosomal organization related to the chromosome's temporal pattern of replication?

Question 33

A trace amount of a radioactively labeled nucleotide is added to a rapidly dividing population of E. coli. After a minute, and again after 30 minutes, nucleic acid is isolated and analyzed for the presence of radioactivity. Explain whether you expect to find radioactivity in small ( 1,000 nucleotide) or large ( 10,000 nucleotide) DNA fragments, or neither, at each time point if the radioactively labeled nucleotide is
a. UTP uniformly labeled witH₃ H (tritium)b. dATP uniformly labeled witH₃ H (tritium)c.
32 P-dATP (dATP where the phosphorus closest to the 5
carbon is radioactive)d.
32 P-UTP (UTP where the phosphorus closest to the 5
carbon is radioactive)e.
32 P-dATP (dATP where the phosphorus furthest from the 5
carbon is radioactive)

Question 34

When the eukaryotic chromosome duplicates, the nucleosome structures must duplicate.
a. How is the synthesis of histones related to the cell cycle?
b. One possibility for the assembly of new nucleosomes on replicated DNA is that it is semiconservative. That is, parental nucleosomes are assembled on one daughter double helix and newly synthesized nucleosomes are synthesized on the other daughter double helix. Is this what happens? If not, what does occur?

Question 35

A mutant Tetrahymena has an altered repeated sequence in its telomeric DNA. What change in the telomerase enzyme would produce this phenotype?

Question 36

What is the evidence that telomere length is regulated in cells, and what are the consequences of the misregulation of telomere length?