Biochemistry

Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto, Jr, Lubert Stryer

Chapter 28

DNA Replication, Repair, and Recombination - all with Video Answers

Educators

CB

Chapter Questions

Problem 1

Activated intermediates. DNA polymerase I, DNA ligase, and topoisomerase I catalyze the formation of phosphodiester bonds. What is the activated intermediate in the linkage reaction catalyzed by each of these enzymes? What is the leaving group?

Lottie Adams

Lottie Adams

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Problem 2

Life in a hot tub. An archaeon (Sulfolobus acidocaldarius) found in acidic hot springs contains a topoisomerase that catalyzes the ATP-driven introduction of positive supercoils into DNA. How might this enzyme be advantageous to this unusual organism?

Lottie Adams

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Problem 3

Which way? Provide a chemical explanation of why DNA synthesis proceeds in a $5^{\prime}$ -to- $3^{\prime}$ direction.

Lottie Adams

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Problem 4

Nucleotide requirement. DNA replication does not take place in the absence of the ribonucleotides ATP, CTP, $\mathrm{GTP},$ and UTP. Propose an explanation.

Lottie Adams

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Problem 5

Close contact. Examination of the structure of DNA polymerases bound to nucleotide analogs reveals that conserved residues come within van der Waals contact of $\mathrm{C}-2^{\prime}$ of the bound nucleotide. What is the potential significance of this interaction?

Lottie Adams

Numerade Educator

Problem 6

Molecular motors in replication.
(a) How fast does template DNA spin (expressed in revolutions per second) at an $E .$ coli replication fork?
(b) What is the velocity of movement (in micrometers per second) of DNA polymerase III holoenzyme relative to the template?

Lottie Adams

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Problem 7

Wound tighter than a drum. Why would replication come to a halt in the absence of topoisomerase II?

Lottie Adams

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Problem 8

The missing link. One form of a plasmid shows a twist of $T w=48$ and a writhe of $W r=3$
(a) What is the linking number?
(b) What would the value of writhe be for a form with twist $T w=50$ if the linking number is the same as that for the preceding form?

Nicholas Sacco

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Problem 9

Telomeres and cancer. Telomerase is not active in most human cells. Some cancer biologists have suggested that activation of the telomerase gene would be a requirement for a cell to become cancerous. Explain why this might be the case.

CB

Christopher Bazell

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Problem 10

Backward? Bacteriophage T7 helicase moves along DNA in the $5^{\prime}-$ to- $3^{\prime}$ direction. Other helicases have been reported to move in the $3^{\prime}-$ to- $5^{\prime}$ direction. Is there any fundamental reason why you would expect helicases to move in one direction or the other?

Lottie Adams

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Problem 11

Nick translation. Suppose that you wish to make a sample of DNA duplex highly radioactive to use as a DNA probe. You have a DNA endonuclease that cleaves the DNA internally to generate $3^{\prime}-\mathrm{OH}$ and $5^{\prime}$ -phosphoryl groups, intact DNA polymerase I, and radioactive dNTPs. Suggest a means for making the DNA radioactive.

Lottie Adams

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Problem 12

Revealing tracks. Suppose that replication is initiated in a medium containing moderately radioactive tritiated thymine. After a few minutes of incubation, the bacteria are transferred to a medium containing highly radioactive tritiated thymidine. Sketch the autoradiographic pattern that would be seen for (a) undirectional replication and (b) bidirectional replication, each from a single origin.

Lottie Adams

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Problem 13

Mutagenic trail. Suppose that the single-stranded RNA from tobacco mosaic virus was treated with a chemical mutagen, that mutants were obtained having serine or leucine instead of proline at a specific position, and that further treatment of these mutants with the same mutagen yielded phenylalanine at this position.
What are the plausible codon assignments for these four amino acids?
FIGURE CANT COPY

Lottie Adams

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Problem 14

Induced spectrum. DNA photolyases convert the energy of light in the near-ultraviolet or visible region $(300-500 \mathrm{nm})$ into chemical energy to break the cyclobutane ring of pyrimidine dimers. In the absence of substrate, these photoreactivating enzymes do not absorb light of wavelengths longer than $300 \mathrm{nm} .$ Why is the substrate-induced absorption band advantageous?

Lottie Adams

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Problem 15

Missing telomerase. Cells lacking telomerase can grow for several cell divisions without obvious defects. However, after more cell divisions, such cells tend to show chromosomes that have fused together. Propose an explanation for the formation of the chromosomes.

Lottie Adams

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Problem 16

I need to unwind. With the assumption that the energy required to break an average base pair in DNA is $10 \mathrm{kJ}$ $\operatorname{mol}^{-1}\left(2.4 \mathrm{kcal} \mathrm{mol}^{-1}\right),$ estimate the maximum number of
base pairs that could be broken per ATP hydrolyzed by a helicase operating under standard conditions.

Lottie Adams

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Problem 17

Triplet oxidation. The oxidation of guanine bases in the context of triplet repeats such as CAGCAGCAG can lead to the expansion of the repeat. Explain.

Lottie Adams

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Problem 18

A revealing analog. AMP-PNP, the $\beta, \gamma$ -imido analog of ATP (p. $833),$ is hydrolyzed very slowly by most ATPases. The addition of AMP-PNP to topoisomerase II and circular DNA leads to the negative supercoiling of a single molecule of DNA per enzyme. DNA remains bound to the enzyme in the presence of this analog. What does this finding reveal about the catalytic mechanism?

Lottie Adams

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Problem 19

Like a ladder. Circular DNA from SV40 virus was isolated and subjected to gel electrophoresis. The results are shown in lane $\mathrm{A}$ (the control) of the adjoining gel patterns. (a) Why does the DNA separate in agarose gel electrophoresis? How does the DNA in each band differ?
The DNA was then incubated with topoisomerase I for
5 minutes and again analyzed by gel electrophoresis with the results shown in lane B.
(b) What types of DNA do the various bands represent?
Another sample of DNA was incubated with topoisomerase I for 30 minutes and again analyzed as shown in lane $\mathrm{C}$.
(c) What is the significance of the fact that more of the DNA is in slower-moving forms?
FIGURE CANT COPY

Lottie Adams

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Problem 20

Ames test. The illustration below shows four petri plates used for the Ames test. A piece of filter paper (white circle in the center of each plate) was soaked in one of four preparations and then placed on a petri plate. The four preparations contained (A) purified water (control), (B) a known mutagen, (C) a chemical whose mutagenicity is under investigation, and (D) the same chemical after treatment with liver homogenate. The number of revertants, visible as colonies on the petri plates, was determined in each case.
(a) What was the purpose of the control plate, which was exposed only to water?
(b) Why was it wise to use a known mutagen in the experimental system?
(c) How would you interpret the results obtained with the experimental compound?
(d) What liver components would you think are responsible for the effects observed in preparation D?
FIGURE CANT COPY

Lottie Adams

Numerade Educator