Chapter 28

1. DNA polymerase I uses deoxyribonucleoside triphosphates; pyrophosphate is the leaving group. DNA ligase uses DNA–adenylate (AMP joined to the 5′-phosphoryl group) as a reaction partner; AMP is the leaving group. Topoisomerase I uses a DNA–tyrosyl intermediate (5′-phosphoryl group linked to the phenolic OH group); the tyrosine residue of the enzyme is the leaving group.

2. Positive supercoiling resists the unwinding of DNA. The melting temperature of DNA increases in proceeding from negatively supercoiled to relaxed to positively supercoiled DNA. Positive supercoiling is probably an adaptation to high temperature.

3. The nucleotides used for DNA synthesis have the triphosphate attached to the 5′-hydroxyl group with free 3′-hydroxyl groups. Such nucleotides can be utilized only for 5′-to-3′ DNA synthesis.

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4. DNA replication requires RNA primers. Without appropriate ribonucleotides, such primers cannot be synthesized.

5. This close contact prevents the incorporation of ribonucleotides rather than 2′-deoxyribonucleotides.

6. (a) 96.2 revolutions per second (1000 nucleotides per second divided by 10.4 nucleotides per turn for B-DNA gives 96.2 rps).

(b) 0.34 µm s−1 (1000 nucleotides per second corresponds to 3400 Å s−1 because the axial distance between nucleotides in B-DNA is 3.4 Å).

7. Eventually, the DNA would become so tightly wound that movement of the replication complex would be energetically impossible.

8. Linking number Lk = Tw + Wr = 48 + 3 = 51. If Tw = 50, then Wr = 1.

9. A hallmark of most cancer cells is prolific cell division, which requires DNA replication. If the telomerase were not activated, the chromosomes would shorten until they became nonfunctional, leading to cell death.

10. No.

11. Treat the DNA briefly with endonuclease to occasionally nick each strand. Add the polymerase with the radioactive dNTPs. At the broken bond, or nick, the polymerase will degrade the existing strand with its 5′ → 3′ exonuclease activity and replace it with a radioactive complementary copy by using its polymerase activity. This reaction scheme is referred to as “nick translation” because the nick is moved, or translated, along the DNA molecule without ever becoming sealed.

12. If replication were unidirectional, tracks with a low grain density at one end and a high grain density at the other end would be seen. On the other hand, if replication were bidirectional, the middle of a track would have a low density, as shown in the diagram below. For E. coli, the grain tracks are denser on both ends than in the middle, indicating that replication is bidirectional.

13. Pro (CCC), Ser (UCC), Leu (CUC), and Phe (UUC). Alternatively, the last base of each of these codons could be U.

14. Potentially deleterious side reactions are prevented. The enzyme itself might be damaged by light if it could be activated by light in the absence of bound DNA harboring a pyrimidine dimer.

15. The free DNA ends that appear in the absence of telomeres are repaired by DNA fusion.

16. The free energy of ATP hydrolysis under standard conditions is −30.5 kJ mol−1 (−7.3 kcal mol−1). In principle, it could be used to break three base pairs.

17. The oxidation of guanine could lead to DNA repair: DNA strand cleavage could allow looping out of the triplet repeat regions and triplet expansion.

18. The release of DNA topoisomerase II after the enzyme has acted on its DNA substrate requires ATP hydrolysis. Negative supercoiling requires only the binding of ATP, not its hydrolysis.

19. (a) Size; the top is relaxed and the bottom is supercoiled DNA. (b) Topoisomers. (c) The DNA is becoming progressively more unwound, or relaxed, and thus slower moving.

20. (a) It was used to determine the number of spontaneous revertants—that is, the background mutation rate.

(b) To firmly establish that the system was working. A known mutagen’s failure to produce revertants would indicate that something was wrong with the experimental system.

(c) The chemical itself has little mutagenic ability but is apparently activated into a mutagen by the liver homogenate.

(d) Cytochrome P450 system.