The base-pairing atoms of thymine are not directly involved in the cyclobutane ring of a pyrimidine dimer, formed by UV irradiation. Why does a pyrimidine dimer stall the replicative DNA polymerase?

For the nucleotide sequence AAC(O⁶-meG)TGCAC, with a damaged (methylated) G residue, what would be the sequence of each strand of the double-stranded DNA in the following situations?

Name three of the common ways in which DNA lesions are incurred. What is required for these DNA lesions to result in a mutation?

Benzo[a]pyrene, the cancer-causing agent in cigarette smoke, is a powerful mutagen. Benzo[a]pyrene itself is relatively harmless, but it is metabolized in the liver to produce active molecules that react covalently with DNA. In an experiment, benzo[a]pyrene is incubated with a mixture of liver enzymes to form its genotoxic metabolites. These metabolites are added to E. coli cells that have a mutation in a gene encoding an enzyme of the serine-synthesizing pathway (i.e., the cells are serine auxotrophs, requiring serine for growth). When the treated cells are grown on serine-containing medium, the results show that the benzo[a]pyrene metabolites kill cells in a dose-dependent manner. When treated and untreated serine-auxotrophic cells are plated separately on serine-free media, the cells treated with benzo[a]pyrene metabolites show a 10- to 100-fold increase in survivors compared with untreated cells. Explain these results.

In the experiment described in Problem 4, some of the untreated serine auxotrophs were able to grow on a medium lacking serine. Why?

In an experiment using S. typhimurium histidine auxotrophs, the cells are grown on a thin layer of agar with nutrient medium that lacks histidine. The culture (∼10⁹ cells) produces ∼13 colonies over a two-day incubation period at 37°C.

What type of mutation is most likely to result from the following lesions (if left unrepaired)?

The human disease known as xeroderma pigmentosum (XP) arises from mutations in at least seven different genes. The resulting deficiencies are generally in enzymes involved in some part of the pathway for nucleotide excision repair. The various types of XP are denoted A through G (XPA, XPB, etc.), with a few additional variants lumped together under the label XP-V. Cultures of fibroblasts from healthy individuals and from patients with XPG are irradiated with UV light. The DNA is isolated and denatured, and the resulting single-stranded DNA is examined by analytical ultracentrifugation.

Describe the most critical difference between global nucleotide excision repair and transcription-coupled repair.

What do base excision repair and repair of an abasic site have in common? How do they differ?

Many eukaryotes have a DNA glycosylase that specifically removes T residues from DNA, but only when they are paired with G. There is no comparable enzyme that removes the G residues from G—T mismatches. Why is it useful for a cell to always repair a G—T mismatch to G≡C rather than to A=T?

A gene is found that has a sequence of 11 contiguous A residues in one strand. Mutations occur at an elevated frequency in this gene, mostly in the region with the repeated A residues. Most of these mutations result in inactivation of the encoded protein, with many amino acids either missing or altered. What type of mutations would account for these observations, and how might they occur?

Many bacteria, including E. coli, are capable of growing under both anaerobic and aerobic conditions. Some mutations are introduced into an E. coli strain that inactivate several enzymes involved in DNA repair. The mutant strain grows normally when kept in an incubator with a 100% nitrogen gas atmosphere. However, the strain dies when exposed to a normal laboratory atmosphere. Why?

In humans, a fetus lacking at least one good copy of a gene encoding any one of dozens of key DNA repair enzymes is usually nonviable. If the fetus has one good copy of the gene and one mutant (inactive) copy, the individual will have a fully functional DNA repair system, but a higher than normal probability of acquiring cancer in middle age. Explain.

In an E. coli cell, DNA polymerase III makes a rare error and inserts a G opposite an A residue at a position 850 bp away from the nearest GATC sequence. The mismatch is accurately repaired by the mismatch repair system. How many phosphodiester bonds derived from deoxynucleotides (dNTPs) are expended in this repair process? ATPs are also used in this process. Which enzymes consume the ATP?

If an oxidative lesion occurs spontaneously in a single-stranded DNA fragment generated on the lagging strand during replication, it is not readily repaired by nucleotide excision repair or base excision repair. Explain why.

O⁶-Methylguanine lesions are repaired directly by transfer of the methyl group to O⁶-methylguanine methyltransferase. A very high level of metabolic energy is invested in this simple methyl transfer reaction. Describe this energy investment.