Chapter 6 tb

The sliding clamp complex encircles the DNA template and binds to DNA polymerase. This helps the polymerase synthesize much longer stretches of DNA without dissociating. While the loading of the clamp only occurs once on the leading strand, it must happen each time a new Okazaki fragment is made on the lagging strand. How does the cell expedite this process?

The cell employs an additional protein in order to make the constant reloading of the sliding clamp on the lagging strand much more efficient. The protein, called the clamp loader, harnesses energy from ATP hydrolysis to lock a sliding clamp complex around the DNA for every successive round of DNA synthesis.

What part of the DNA replication process would be most directly affected if a strain of bacteria lacking DNA ligase were used to make the cell extracts?

lagging-strand completion

What part of the DNA replication process would be most directly affected if a strain of bacteria lacking the exonuclease activity of DNA polymerase were used to make the cell extracts?

lagging-strand completion

The DNA duplex consists of two long covalent polymers wrapped around each other many times over their entire length. The separation of the DNA strands for replication causes the strands to be "overwound" in front of the replication fork. How does the cell relieve the torsional stress created along the DNA duplex during replication?

Topoisomerases break the covalent bonds of the backbone allowing the local unwinding of DNA ahead of the replication fork.

The classic experiments conducted by Meselson and Stahl demonstrated that DNA replication is accomplished by employing a ________________ mechanism.

semiconservative

The process of DNA replication requires that each of the parental DNA strands be used as a ___________________ to produce a duplicate of the opposing strand.

template

Initiator proteins bind to replication origins and disrupt hydrogen bonds between the two DNA strands being copied. Which of the factors below does not contribute to the relative ease of strand separation by initiator proteins?

the reaction can occur at room temperature

How does the total number of replication origins in bacterial cells compare with the number of origins in human cells?

1 versus 10,000

The chromatin structure in eukaryotic cells is much more complicated than that observed in prokaryotic cells. This is thought to be the reason that DNA replication occurs much faster in prokaryotes. How much faster is it?

10×

How many replication forks are formed when an origin of replication is opened?

2

What part of the DNA replication process would be most directly affected if a strain of bacteria lacking single-strand binding protein were used to make the cell extracts?

Okazaki fragment synthesis

Indicate whether the following statements are true or false. If a statement is false, explain why it is false. A. The repair polymerase is the enzyme that proofreads the newly synthesized strands to ensure the accuracy of DNA replication. B. There is a single enzyme that degrades the RNA primers and lays down the corresponding DNA sequence behind it. C. DNA ligase is required to seal the sugar-phosphate backbone between all the DNA fragments on the lagging strand. D. The repair polymerase does not require the aid of the sliding clamp, because it is only synthesizing DNA over very short stretches.

A. False. The repair polymerase is used to fill in the spaces left vacant after the RNA primers are degraded. B. False. This is a two-step process that requires two different enzymes. First, a nuclease removes the RNA primers. Then, the repair polymerase fills in the complementary DNA sequence. C. True. D. True.

Indicate whether the following statements are true or false. If a statement is false, explain why it is false. A. When DNA is being replicated inside a cell, local heating occurs, allowing the two strands to separate. B. DNA replication origins are typically rich in G-C base pairs. C. Meselson and Stahl ruled out the dispersive model for DNA replication. D. DNA replication is a bidirectional process that is initiated at multiple locations along chromosomes in eukaryotic cells.

A. False. The two strands do need to separate for replication to occur, but this is accomplished by the binding of initiator proteins at the origin of replication. B. False. DNA replication origins are typically rich in A-T base pairs, which are held together by only two hydrogen bonds (instead of three for C-G base pairs), making it easier to separate the strands at these sites. C. True. D. True.

Answer the following questions about DNA replication. A. On a DNA strand that is being synthesized, which end is growing—the 3′ end, the 5′ end, or both ends? Explain your answer. B. On a DNA strand that is being used as a template, where is the copying occurring relative to the replication origin—3′ of the origin, 5′, or both?

A. The 3′ end. DNA polymerase can add nucleotides only to the 3′-OH end of a nucleic acid chain. B. Both, as a result of the bidirectional nature of chromosomal replication.

Meselson and Stahl grew cells in media that contained different isotopes of nitrogen (15N and 14N) so that the DNA molecules produced from these different isotopes could be distinguished by mass. A. Explain how "light" DNA was separated from "heavy" DNA in the Meselson and Stahl experiments. B. Describe the three existing models for DNA replication when these studies were begun, and explain how one of them was ruled out definitively by the experiment you described for part A. C. What experimental result eliminated the dispersive model of DNA replication?

A. The DNA samples collected were placed into centrifuge tubes containing cesium chloride. After high-speed centrifugation for 2 days, the heavy and light DNA products were separated by density. B. The three models were conservative, semiconservative, and dispersive. The conservative model suggested a mechanism by which the original parental strands stayed together after replication and the daughter duplex was made entirely of newly synthesized DNA. The semiconservative model proposed that the two DNA duplexes produced during replication were hybrid molecules, each having one of the parental strands and one of the newly synthesized strands. The dispersive model predicted that the new DNA duplexes each contained segments of parental and daughter strands all along the molecule. The conservative model was ruled out by the density-gradient experiments. C. The dispersive model was ruled out by using heat to denature the DNA duplexes and then comparing the densities of the single-stranded DNA. If the dispersive model had been correct, individual strands should have had an intermediate density. However, this was not the case; only heavy strands and light strands were observed, which convincingly supported the semiconservative model for DNA replication.

Researchers have isolated a mutant strain of E. coli that carries a temperature-sensitive variant of the enzyme DNA ligase. At the permissive temperature, the mutant cells grow just as well as the wild-type cells. At the nonpermissive temperature, all of the cells in the culture tube die within 2 hours. DNA from mutant cells grown at the nonpermissive temperature for 30 minutes is compared with the DNA isolated from cells grown at the permissive temperature. The results are shown in Figure Q6-33, where DNA molecules have been separated by size by means of electrophoresis (P, permissive; NP, nonpermissive). Explain the appearance of a distinct band with a size of 200 base pairs (bp) in the sample collected at the nonpermissive temperature.

DNA ligase has an important role in DNA replication. After Okazaki fragments are synthesized, they must be ligated (covalently connected) to each other so that they finally form one continuous strand. At the nonpermissive temperature this does not happen, and although there may be a range of fragments, the notable band at 200 base pairs is the typical size of an individual Okazaki fragment.

If the genome of the bacterium E. coli requires about 20 minutes to replicate itself, how can the genome of the fruit fly Drosophila be replicated in only 3 minutes?

Drosophila DNA contains more origins of replication than E. coli DNA. Explanation: Bacteria have one origin of replication, and Drosophila has many.The Drosophila genome is bigger than the E. coli genome. Eukaryotic polymerases are not faster than prokaryotic polymerases.

Which of the following statements correctly explains what it means for DNA replication to be bidirectional?

The replication forks formed at the origin move in opposite directions.

Which of the following statements about sequence proofreading during DNA replication is false?

The exonuclease activity cleaves DNA in the 5′-to-3′ direction.

Which of the following statements is true with respect to this in vitro replication system?

The leading and lagging strands compose one half of each newly synthesized DNA strand. Leading and lagging strands are synthesized bidirectionally from the replication origin, and are joined together by DNA ligase where the two replication forks meet at the termination site. The replication fork is bidirectional. The origin is a specialized sequence where initiator proteins bind and open the DNA so that the DNA replication machinery can assemble. The daughter DNA molecules will be same size as the original plasmid (and each other).

Use your knowledge of how a new strand of DNA is synthesized to explain why DNA replication must occur in the 5′-to-3′ direction. In other words, what would be the consequences of 3′-to-5′ strand elongation?

There would be several detrimental consequences to 3′-to-5′ strand elongation. One of those most directly linked to the processes of DNA replication involves synthesis of the lagging strand. After the RNA primers are degraded, the DNA segments remaining will have 5′ ends with a single phosphate group. The incoming nucleotide will have a 3′-OH group. Without the energy provided by the release of PPi from the 5′ end, the process of elongation would no longer be energetically favorable.

You prepare bacterial cell extracts by lysing the cells and removing insoluble debris via centrifugation. These extracts provide the proteins required for DNA replication. Your DNA template is a small, double-stranded circular piece of DNA (a plasmid) with a single origin of replication and a single replication termination site. The termination site is on the opposite side of the plasmid from the origin. In addition to the extracts and the plasmid DNA, are there any additional materials you should add to this in vitro replication system? Explain your answer.

You will probably add exogenous nucleoside triphosphates to serve as the building blocks needed to make new strands of DNA. Although these monomers will be present in the extracts, they will be present at lower concentrations than are normally found inside the cell. They may also be subject to hydrolysis, and the nucleoside diphosphates that are the products of this hydrolysis are not usable substrates for DNA replication. For both of these reasons, it is important to add excess nucleotides to the reaction mixture for efficient DNA replication to occur.

DNA replication is considered semiconservative because ____________________________.

each daughter DNA molecule consists of one strand from the parent DNA molecule and one new strand.

DNA polymerase catalyzes the joining of a nucleotide to a growing DNA strand. What prevents this enzyme from catalyzing the reverse reaction?

hydrolysis of pyrophosphate (PPi) to inorganic phosphate (Pi) + Pi

What part of the DNA replication process would be most directly affected if a strain of bacteria lacking primase were used to make the cell extracts?

initiation of DNA synthesis DNA synthesis cannot begin without the initial primers. Lagging-strand synthesis requires continual use of RNA primers for discontinuous replication to occur.

What part of the DNA replication process would be most directly affected if a strain of bacteria lacking helicase were used to make the cell extracts?

initiation of DNA synthesis Helicase unwinds the two DNA template strands, replication of both strands depends upon the activity of helicase at the time of initiation.