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?

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?

Which way? Provide a chemical explanation of why DNA synthesis proceeds in a 5′-to-3′ direction.

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

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

Molecular motors in replication.

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

The missing link. One form of a plasmid shows a twist of Tw = 48 and a writhe of Wr = 3.

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.

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

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′-OH and 5′-phosphoryl groups, intact DNA polymerase I, and radioactive dNTPs. Suggest a means for making the DNA radioactive.

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.

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?

Induced spectrum. DNA photolyases convert the energy of light in the near-ultraviolet or visible region (300–500 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 nm. Why is the substrate-induced absorption band advantageous?

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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.

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

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.

A revealing analog. AMP-PNP, the β,γ-imido analog of ATP, 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?

Like a ladder. Circular DNA from SV40 virus was isolated and subjected to gel electrophoresis. The results are shown in lane A (the control) of the adjoining gel patterns.

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.