Shown below are cartoon drawings of four molecules observed in the electron microscope after cutting a circular plasmid once with a restriction enzyme. Does this plasmid replicate bidirectionally or unidirectionally from the origin? Explain. Order the molecules by time of replication, from the earliest to the latest.

A DNA polymerase cannot synthesize a DNA strand de novo from a mixture of deoxynucleotide triphosphates. Explain the role of the primer and template in the reaction promoted by this enzyme.

Arthur Kornberg and his colleagues incubated an extract of E. coli with a mixture of dATP, dTTP, dGTP, and dCTP. Only the dTTP was labeled with ³²P in the α-phosphate group. After incubation, the mixture was treated with trichloroacetic acid to precipitate DNA, but not the dNTPs. The precipitate was collected, and the extent of [³²P]dTMP incorporated into DNA was determined.

Provide at least two observations suggesting that Pol I is not the chromosomal replicase.

You are characterizing a new DNA polymerase. When the enzyme is incubated with [³²P]DNA and no dNTPs, you observe the release of [³²P]dNMPs. This release is prevented by adding unlabeled dNTPs. Explain the reactions that most likely underlie these observations. What would you expect to observe if you added pyrophosphate instead of dNTPs?

Explain how the enzymes that join together Okazaki fragments ensure that all the RNA is removed from the ends of fragments before they are sealed.

A key step in the initiation of replication in bacteria is to load the DnaB helicase onto the DNA at the replication origin. What proteins function at the replication origin during initiation prior to the loading of DnaB?

A DNA fragment containing an origin has been identified, using the recombinant plasmid approach (see this chapter’s How We Know section). The origin contains six short palindromes: four A sites of similar sequence, and two B sites unrelated to the A sites.

Five DNA polymerases have been identified in E. coli. Two of them—DNA polymerases IV and V—lack a 3′→5′ exonuclease activity. What is the function of the 3′→5′ exonuclease activity present in many DNA polymerases? How does the absence of such an activity in DNA polymerases IV and V make sense in the context of the cellular function of these enzymes?

In eukaryotes, DNA replication occurs in the S phase of the cell cycle. However, important preparatory events occur in the G₁ phase, prior to S phase. Describe these events and the status of eukaryotic replication origins as a cell enters S phase.

Eukaryotic origins are tightly regulated so as not to fire more than once during S phase. Explain the key points of regulation of this process in eukaryotic cells.

Explain the role of the τ subunit of Pol III.

The AAA+ ATPases are the wrenches and crowbars of DNA metabolism. Briefly indicate what is accomplished when ATP is hydrolyzed by each of the following AAA+ ATPases: DnaA, DnaC, and the γ or τ subunits of Pol III.

The replication termini (Ter sites) on the E. coli chromosome are oriented as shown below. Briefly describe what would happen in one round of replication if the orientations of all the Ter sites in the chromosome were reversed.

The enzyme telomerase is considered a reverse transcriptase, synthesizing DNA on an RNA template. What is the source of the RNA used in the synthesis of telomeres, where is it located, and how many nucleotides of the RNA are actually used as template for DNA synthesis?