Problem 2
Heavy Isotope Analysis of DNA Replication A researcher switches a culture of \(E\). coli growing in a medium containing \({ }^{15} \mathrm{NH}_{4} \mathrm{Cl}\) to a medium containing \({ }^{14} \mathrm{NH}_{4} \mathrm{Cl}\) for three generations (an eightfold increase in population). What is the molar ratio of hybrid DNA \(\left({ }^{15} \mathrm{~N}^{-14} \mathrm{~N}\right)\) to light DNA \(\left({ }^{14} \mathrm{~N}^{-14} \mathrm{~N}\right)\) at this point?
Problem 4
Base Composition of DNAs Made from Single-Stranded Templates Predict the base composition of the total DNA synthesized by DNA polymerase on templates provided by an equimolar mixture of the two complementary strands of bacteriophage \(\phi \mathrm{X} 174 \mathrm{DNA}\) (a circular DNA molecule). The base composition of one strand is A, \(24.7 \% ; \mathrm{G}, 24.1 \% ; \mathrm{C}\), \(18.5 \%\); and \(\mathrm{T}, 32.7 \%\). What assumption is necessary to answer this problem?
Problem 5
DNA Replication Kornberg and his colleagues incubated soluble extracts of \(E\). coli with a mixture of dATP, dTTP, dGTP, and dCTP, all labeled with \({ }^{32} \mathrm{P}\) in the \(a\)-phosphate group. After a time, they treated the incubation mixture with trichloroacetic acid, which precipitates the DNA but not the nucleotide precursors. They then collected the precipitate and determined the extent of precursor incorporation into DNA from the amount of radioactivity present in the precipitate. a. If any one of the four nucleotide precursors were omitted from the incubation mixture, would radioactivity be found in the precipitate? Explain. b. Would \({ }^{32} \mathrm{P}\) be incorporated into the DNA if only dTTP were labeled? Explain. c. Would radioactivity be found in the precipitate if \({ }^{32} \mathrm{P}\) labeled the \(\beta\) phosphate or \(\gamma\) phosphate rather than the \(a\) phosphate of the deoxyribonucleotides? Explain.
Problem 6
The Chemistry of DNA Replication All DNA polymerases synthesize new DNA strands in the \(5^{\prime} \rightarrow 3^{\prime}\) direction. In some respects, replication of the antiparallel strands of duplex DNA would be simpler if there were also a second type of polymerase, one that synthesized DNA in the \(3^{\prime} \rightarrow 5^{\prime}\) direction. The two types of polymerase could, in principle, coordinate DNA synthesis without the complicated mechanics required for lagging strand replication. However, no such \(3^{\prime} \rightarrow 5^{\prime}\)-synthesizing enzyme has been found. Suggest two possible mechanisms for \(3^{\prime} \rightarrow 5^{\prime}\) DNA synthesis. Pyrophosphate should be one product of both proposed reactions. Could one or both mechanisms be supported in a cell? Why or why not? (Hint: You may suggest the use of DNA precursors not actually present in extant cells.)
Problem 7
Activities of DNA Polymerases You are characterizing a new DNA polymerase. When you incubate the enzyme with \({ }^{32} \mathrm{P}\)-labeled DNA and no dNTPs, you observe the release of \(\left[{ }^{32} \mathrm{P}\right] \mathrm{dNMPs}\). The addition of unlabeled dNTPs prevents this release. Explain the reactions that most likely underlie these observations. What would you expect to observe if you added pyrophosphate instead of dNTPs?
Problem 8
Leading and Lagging Strands Prepare a table that lists the names and compares the functions of the precursors, enzymes, and other proteins needed to make the leading strand versus the lagging strand during DNA replication in \(E\). coli.
Problem 9
Function of DNA Ligase Some \(E\). coli mutants contain defective DNA ligase. When researchers expose these mutants to \({ }^{3} \mathrm{H}\)-labeled thymine and then sediment the DNA produced on an alkaline sucrose density gradient, two radioactive bands appear. One corresponds to a high molecular weight fraction, the other to a low molecular weight fraction. Explain.
Problem 10
Fidelity of Replication of DNA What factors promote the fidelity of replication during synthesis of the leading strand of DNA? Would you expect the lagging strand to be made with the same fidelity? Give reasons for your answers.
Problem 12
The Ames Test In a nutrient medium that lacks histidine, a thin layer of agar containing \(\sim 10^{9}\) Salmonella typhimurium histidine auxotrophs (mutant cells that require histidine to survive) produces \(\sim 13\) colonies over a two-day incubation period at \(37^{\circ} \mathrm{C}\) (see Eig \(25-19\) ). How do these colonies arise in the absence of histidine? When investigators repeat the experiment in the presence of \(0.4 \mu \mathrm{g}\) of 2 -aminoanthracene, the number of colonies produced over two days exceeds 10,000 . What does this indicate about 2-aminoanthracene? What can you surmise about its carcinogenicity?
Problem 13
DNA Repair Mechanisms Vertebrate and plant cells often methylate cytosine in DNA to form 5-methylcytosine (see \(\underline{\text { Fig. }}\) 8-5a). In these same cells, a specialized repair system recognizes \(\mathrm{G}-\mathrm{T}\) mismatches and repairs them to \(\mathrm{G} \equiv \mathrm{C}\) base pairs. How might this repair system be advantageous to the cell? (Explain in terms of the presence of 5-methylcytosine in the DNA.)
