1.  Guide and starting point. Define template and primer as they relate to DNA synthesis. ✓ 3

2.  Cooperation among polymerases. Explain why DNA synthesis depends on RNA synthesis. ✓ 4

3.  One from many. What is an Okazaki fragment? ✓ 4

4.  Foremost and reluctant. Distinguish between the leading and the lagging strands in DNA synthesis. ✓ 4

5.  Like Homer and Marge. Match each term with its description.

6.  Which way? Explain, on the basis of nucleotide structure, why DNA synthesis proceeds in the 5′-to-3 direction. ✓ 4

7.  Strength in numbers. For long double-stranded DNA molecules, the rate of spontaneous strand separation is negligibly low under physiological conditions despite the fact that only weak reversible bonds hold the strands together. Explain.

8.  Only one oar in the water. DNA replication in E. coli begins at a unique site on the circular chromosome called the origin of replication and proceeds in both directions as illustrated in part A.

What would be the effect of a mutation that yielded only one functional fork per origin of replication site (part B)? ✓ 4

9.  Wound tighter than a drum. Why would replication come to a halt in the absence of topoisomerase II? ✓ 3

10.  And just like that, poof. He’s gone. Telomerase is not active in most human cells. Some cancer biologists have suggested that the activation of the telomerase gene would be a requirement for a cell to become cancerous. Explain. ✓ 4

11.  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. ✓ 3

12.  The terminator. (a) What would be the effect of adding dideoxyadenosine triphosphate (ddATP) to a DNA-replication reaction in large excess of deoxyadenosine triphosphate (dATP)? (b) What would happen if ddATP were added at 10% of the concentration of dATP?

13.  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? ✓ 3

14.  Like a ladder. Circular DNA from SV40 virus was isolated and subjected to gel electrophoresis. The results are shown below in lane A (the control) of the electrophoretic patterns. ✓ 3



(a) Why does the DNA separate in agarose gel electrophoresis? How does the DNA in each band differ?

The DNA was then incubated with topoisomerase I for 5 minutes and again analyzed by gel electrophoresis with the results shown in lane B.

(b) What types of DNA do the various bands represent? Another sample of DNA was incubated with topoisomerase I for 30 minutes and again analyzed as shown in lane C.

(c) What is the significance of the fact that more of the DNA is in slower-moving forms?

15.  Don’t rush it. The accuracy rate for DNA polymerase falls if the next nucleotide to be polymerized is present in high concentration. Conversely, the accuracy rate rises if the next nucleotide is present in low concentration, a phenomenon termed the “next nucleotide effect.” Suggest an explanation for this effect. ✓ 3

16.  Fast flying fork. (a) How fast does template DNA spin (expressed in revolutions per second) at an E. coli replication fork? (b) What is the velocity of movement (in micrometers per second) of DNA polymerase III holoenzyme relative to the template?

17.  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. ✓ 3