Comprehension Questions

1. What is semiconservative replication?

* 2. How did Meselson and Stahl demonstrate that replication in E. coli takes place in a semiconservative manner?

* 3. Draw a molecule of DNA undergoing theta replication. On your drawing, identify (1) origin, (2) polarity (5' and 3' ends) of all template strands and newly synthesized strands,

(3) leading and lagging strands, (4) Okazaki fragments, and (5) location of primers.

4. Draw a molecule of DNA undergoing rolling-circle replication. On your drawing, identify (1) origin,

(2) polarity (5' and 3' ends) of all template and newly synthesized strands, (3) leading and lagging strands,

(4) Okazaki fragments, and (5) location of primers.

5. Draw a molecule of DNA undergoing eukaryotic linear replication. On your drawing, identify (1) origin; (2) polarity (5' and 3' ends) of all template and newly synthesized strands, (3) leading and lagging strands, (4) Okazaki fragments, and (5) location of primers.

6. What are three major requirements of replication?

* 7. What substrates are used in the DNA synthesis reaction?

8. List the different proteins and enzymes taking part in bacterial replication. Give the function of each in the replication process.

9. What similarities and differences exist in the enzymatic activities of DNA polymerases I, II, and III? What is the function of each type of DNA polymerase in bacterial cells?

*10. Why is primase required for replication?

11. What three mechanisms ensure the accuracy of replication in bacteria?

12. How does replication licensing ensure that DNA is replicated only once at each origin per cell cycle?

*13. In what ways is eukaryotic replication similar to bacterial replication, and in what ways is it different?

14. Outline in words and pictures how telomeres at the end of eukaryotic chromosomes are replicated.

15. Briefly outline with diagrams the Holliday model of homologous recombination.

*16. What are some of the enzymes taking part in recombination in E. coli and what roles do they play?

APPLICATION QUESTIONS AND PROBLEMS]_

*17. Suppose a future scientist explores a distant planet and discovers a novel form of double-stranded nucleic acid. When this nucleic acid is exposed to DNA polymerases from E. coli, replication takes place continuously on both strands. What conclusion can you make about the structure of this novel nucleic acid?

*18. Phosphorus is required to synthesize the deoxyribonucleoside triphosphates used in DNA replication. A geneticist grows some E. coli in a medium containing nonradioactive phosphorous for many generations. A sample of the bacteria is then transferred to a medium that contains a radioactive isotope of phosphorus (32P). Samples of the bacteria are removed immediately after the transfer and after one and two rounds of replication. What will be the distribution of radioactivity in the DNA of the bacteria in each sample? Will radioactivity be detected in neither, one, or both strands of the DNA?

19. A line of mouse cells is grown for many generations in a medium with 15N. Cells in G1 are then switched to a new medium that contains 14N. Draw a pair of homologous chromosomes from these cells at the following stages, showing the two strands of DNA molecules found in the chromosomes. Use different colors to represent strands with 14N and 15N.

(a) Cells in G:, before switching to medium with 14N

(b) Cells in G2, after switching to medium with 14N

(c) Cells in anaphase of mitosis, after switching to medium with 14N

(d) Cells in metaphase I of meiosis, after switching to medium with 14N

(e) Cells in anaphase II of meiosis, after switching to medium with 14N

*20. A circular molecule of DNA contains 1 million base pairs. If DNA synthesis at a replication fork occurs at a rate of 100,000 nucleotides per minute, how long will theta replication require to completely replicate the molecule, assuming that theta replication is bidirectional? How long will replication of this circular chromosome take by rolling-circle replication? Ignore replication of the displaced strand in rolling-circle replication.

21. A bacterium synthesizes DNA at each replication fork at a rate of 1000 nucleotides per second. If this bacterium completely replicates its circular chromosome by theta replication in 30 minutes, how many base pairs of DNA will its chromosome contain?

*22. The following diagram represents a DNA molecule that is undergoing replication. Draw in the strands of newly synthesized DNA and identify the following:

(a) Polarity of newly synthesized strands

(b) Leading and lagging strands

(c) Okazaki fragments

(d) RNA primers

*23. What would be the effect on DNA replication of mutations that destroyed each of the following activities in DNA polymerase I?

Origin

Unwinding

Origin

(a) 3':5' exonuclease activity

(b) 5':3' exonuclease activity

(c) 5':3' polymerase activity

Unwinding

CHALLENGE QUESTIONS]_

24. Conditional mutations express their mutant phenotype only under certain conditions (the restrictive conditions) and express the normal phenotype under other conditions (the permissive conditions). One type of conditional mutation is a temperature-sensitive mutation, which expresses the mutant phenotype only at certain temperatures.

Strains of E. coli have been isolated that contain temperature-sensitive mutations in the genes encoding different components of the replication machinery. In each of these strains, the protein produced by the mutated gene is nonfunctional under the restrictive conditions. These strains are grown under permissive conditions and then abruptly switched to the restrictive condition. After one round of replication under the restrictive condition, the DNA from each strain is isolated and analyzed. What would you predict to see in the DNA isolated from each strain in the following list?

Temperature-sensitive mutation in gene encoding:

(a) DNA ligase

(b) DNA polymerase I

(c) DNA polymerase III

(d) Primase

(e) Initiator protein

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