8. DNA Replication

Distinguish between

(a) unidirectional and bidirectional synthesis, and

(b) continuous and discontinuous synthesis of DNA.

Diagram a replication fork in bacterial DNA and label the following structures or molecules.

a. DNA pol III

b. Helicase

c. RNA primer

d. Origin of replication

e. leading strand (label its polarity)

f. DNA pol I

g. Topoisomerase

h. SSB protein

i. Lagging strand (label its polarity)

j. Primase

k. Okazaki fragment

List the proteins that unwind DNA during in vivo DNA synthesis. How do they function?

Define and indicate the significance of

(a) Okazaki fragments,

(b) DNA ligase, and

(c) primer RNA during DNA replication.

Outline the current model for DNA synthesis.

Why is DNA synthesis expected to be more complex in eukaryotes than in bacteria? How is DNA synthesis similar in the two types of organisms?

Suppose that E. coli synthesizes DNA at a rate of 100,000 nucleotides per minute and takes 40 minutes to replicate its chromosome.

(a) How many base pairs are present in the entire E. coli chromosome?

(b) What is the physical length of the chromosome in its helical configuration—that is, what is the circumference of the chromosome if it were opened into a circle?

Several temperature-sensitive mutant strains of E. coli display the following characteristics. Predict what enzyme or function is being affected by each mutation.

Supercoiled strands remain after replication, which is never completed.

Several temperature-sensitive mutant strains of E. coli display the following characteristics. Predict what enzyme or function is being affected by each mutation.

Synthesis is very slow.

Several temperature-sensitive mutant strains of E. coli display the following characteristics. Predict what enzyme or function is being affected by each mutation.

No initiation occurs.

Several temperature-sensitive mutant strains of E. coli display the following characteristics. Predict what enzyme or function is being affected by each mutation.

Okazaki fragments accumulate, and DNA synthesis is never completed.

Several temperature-sensitive mutant strains of E. coli display the following characteristics. Predict what enzyme or function is being affected by each mutation.

Newly synthesized DNA contains many mismatched base pairs.

DNA supercoiling, which occurs when coiling tension is generated ahead of the replication fork, is relieved by DNA gyrase. Supercoiling may also be involved in transcription regulation. Researchers discovered that enhancers operating over a long distance (2500 bp) are dependent on DNA supercoiling, while enhancers operating over shorter distances (110 bp) are not so dependent [Liu et al. (2001). Proc. Natl. Acad. Sci. USA 98:14,883–14,888]. Using a diagram, suggest a way in which supercoiling may positively influence enhancer activity over long distances.

Raymond Rodriguez and colleagues demonstrated conclusively that DNA replication in E. coli is bidirectional. Explain why locating the origin of replication on one side of the circular chromosomes and the terminus of replication on the opposite side of the chromosome supported this conclusion.

While many commonly used antibiotics interfere with protein synthesis or cell wall formation, clorobiocin, one of several antibiotics in the aminocoumarin class, inhibits the activity of bacterial DNA gyrase. Similar drugs have been tested as treatments for human cancer. How might such drugs be effective against bacteria as well as cancer?