Chapter 22

Describe an experimental approach to determining the processivity of a DNA polymerase (i.e., the number of nucleotides incorporated per chain per polymerase binding event).

Adenylate cyclase, which synthesizes cyclic AMP from ATP, requires two metal ions, and the enzyme has the same constellation of amino acid residues in the active site as does DNA polymerase I. In what sense is the adenylate cyclase reaction similar to that of DNA polymerase, and in what sense is it different?

Deoxyadenylate residues in DNA undergo deamination fairly readily, as do deoxycytidylate residues. (a) What is the product of dAMP deamination? (b) The deamination product is known to base-pair with \(\mathrm{A}, \mathrm{C}\), or \(\mathrm{T}\). What would be the genetic consequences if this deaminated site in DNA were not repaired and if it paired with \(C\) on the next round of replication?

The E. coli chromosome is \(1.28 \mathrm{~mm}\) long. Under optimal conditions, the chromosome is replicated in 40 minutes. (a) What is the distance traversed by one replication fork in 1 minute? (b) If replicating DNA is in the B form (10.4 base pairs per turn), how many nucleotides are incorporated in 1 minute in one replication fork? (c) If cultured human cells (such as HeLa cells) replicate \(1.2 \mathrm{~m}\) of DNA during a five-hour \(\mathrm{S}\) phase and at a rate of fork movement one-tenth of that seen in \(E\). coli, how many origins of replication must the cells contain? (d) What is the average distance, in kilobase pairs, between these origins?

DNA ligase has the ability to relax supercoiled circular DNA in the presence of AMP but not in its absence. (a) What is the mechanism of this reaction, and why does it depend on AMP? (b) How could you determine that supercoiled DNA had in fact been relaxed?

Suppose that a replicative DNA polymerase had its \(3^{\prime}\) exonuclease site \(1.5 \mathrm{~nm}\) from the polymerase site, rather than the \(3.0 \mathrm{~nm}\) seen in Klenow fragment. How would this change affect the fidelity of the enzyme? Why? Describe an additional change that could give this enzyme the same fidelity as Klenow fragment while retaining the \(1.5\)-nm inter-site distance.

Although DNA polymerases require both a template and a primer, the following single-stranded polynucleotide was found to serve as a substrate for DNA polymerase in the absence of any additional DNA. 3' HO-ATGGGCTCATAGCCGGAGCCCTAACC- GTAGACCACGAATAGCATTAGG-p \(5^{\prime}\) What is the structure of the product of this reaction?

The \(3^{\prime}\)-exonuclease activity of \(E\). coli DNA polymerase I was found to show no discrimination between correctly and incorrectly base-paired nucleotides at the \(3^{\prime}\)-terminus; properly and improperly base-paired nucleotides are cleaved at equal rates there. How can this observation be reconciled with the fact that the \(3^{\prime}\)-exonuclease activity increases the accuracy with which template DNA is copied?

What information would you need to tell whether the leading and lagging strands in yeast are replicated with equal fidelity?

It takes 40 minutes to completely replicate the \(E\) coli chromosome, even in an optimally nourished cell. However, bacterial cells can divide as frequently as every 20 minutes. How can cells divide more rapidly, apparently, than their DNA can be copied?

5-Bromouracil (BU) resembles thymine sufficiently that BU base-pairs readily with adenine in a DNA helix, and it can readily substitute for thymine in DNA replication. However, its electron distribution allows it to resemble cytosine sufficiently that BU can pair with guanine. Show how a few rounds of replication in the presence of \(B U\) could convert an \(A-T\) base pair to \(\mathrm{G}-\mathrm{C}\), and identify the products of each round of replication.

The oxidation of a DNA-guanine residue to 8-oxoguanine (8-oxoG) is mutagenic because 8 -oxoG pairs readily with adenine in the next round of replication. Show a pathway beginning with the oxidation of \(\mathrm{G}\) in a \(\mathrm{G}-\mathrm{C}\) base pair and leading to a mutant base pair, and identify the products of each round of replication.

In mammalian cells, genes that are expressed in a particular cell are reported to undergo replication during the first half of \(S\) phase, and genes not expressed in that cell are replicated in the latter half of S phase. Briefly describe an experiment that could lead to this conclusion. You might consider approaches that involve 5 -bromouracil incorporation.

DNA precursor imbalances are mutagenic. For example, if dGTP accumulates, it can compete with dATP for incorporation opposite dTMP in the template, leading to a transition mutation. Investigators have shown that a modest balanced increase in all four dNTPs, three- or fourfold, stimulated mutagenesis out of proportion to the dNTP pool change. Describe a mechanism by which this could occur.

In the mitochondrial disease MNGIE (see page 709) a deficiency of thymidine phosphorylase causes dTTP to accumulate in mitochondria. Describe the mechanism by which this occurs. A secondary effect is depletion of mitochondrial dCTP pools. Describe a plausible mechanism for this effect.

A major difficulty in preparing vaccines against viral diseases is the rapidity with which the virus mutates to vaccine resistance. What is the likely mechanism for this hypermutability? Would you expect this problem to affect DNA and RNA viruses equally? Explain.

Bacteriophage T4 mutants defective in genetic recombination usually show defective DNA replication as well. What specific defect might you expect to see? Describe a plausible explanation for this effect.