life11e_ch13

DNA polymerases add nucleotides to the growing chain

DNA replication begins with the binding of a large protein complex (the pre-replication complex) to a specific site on the DNA molecule. This complex contains several different proteins, including the enzyme DNA polymerase, which catalyzes the addition of nucleotides as the new DNA chain grows. All chromosomes have at least one region called the origin of replication (ori), to which the pre-replication complex binds. Binding occurs when proteins in the complex recognize specific DNA sequences within the ori.

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investigating life

The Meselson–Stahl Experiment

experiment

Original Paper: Meselson, M. and F. Stahl. 1958. The replication of DNA in Escherichia coli. Proceedings of the National Academy of Sciences USA 44: 671–682.

A centrifuge was used to separate DNA molecules labeled with isotopes of different densities. This experiment revealed a pattern that supports the semiconservative model of DNA replication.

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work with the data

The Meselson–Stahl experiment has been called one of the “most beautiful experiments” in biology because of its essential simplicity. Meselson and Stahl used density gradients to examine how DNA molecules replicate, as illustrated in the experimental results shown in Figure A. The peaks on the drawing are proportional to the amount of DNA.

Figure B shows results of the experiment after each of four generations. Each sample contained the same number of bacteria, so the total amount of DNA in each panel was the same.

QUESTIONS

Question 1

Use the heights of the peaks to estimate the percent of total DNA that was heavy, intermediate, and light at each generational stage. Create a table summarizing these calculations and discuss whether they support the authors’ conclusions.

These data fit the semiconservative model of DNA replication because the heavy strands were templates for new light strands; after one round of replication, all the DNA had one heavy (original) strand and one light (newly made) strand, and so was intermediate in weight.

Generation	Percent intermediate DNA	Percent light DNA
1	100	0
2	50	50
3	25	75
4	12.5	87.5

Question 2

What would the data look like if the bacteria had been allowed to divide for three more generations?

After seven generations there would be about 1.5 percent intermediate DNA and 98.5 percent light DNA.

Question 3

If Meselson and Stahl had done their experiment starting with light DNA and then added ¹⁵N for succeeding generations, what would the bands look like?

In the first generation, the bands would be the same as in Figure B: all intermediate. In the second generation, 1/2 would be intermediate and 1/2 heavy. In the third generation, 1/4 would be intermediate and 3/4 heavy. In the fourth generation, 1/8 would be intermediate and 7/8 heavy.

Question 4

What would the data look like if conservative replication were the correct model? What would the data look like if dispersive replication were correct?

In a conservative model, the first generation would be 1/2 heavy, 1/2 light; the second generation 1/4 heavy, 3/4 light; the third generation 1/8 heavy, 7/8 light; and the fourth generation 1/16 heavy, 15/16 light. In a dispersive model, the first generation would be all intermediate; the second generation all half-way between intermediate and light; the third generation all half-way between the second generation peak and light; and the fourth generation all half-way between the third generation peak and light.

A similar work with the data exercise may be assigned in LaunchPad.

ORIGINS OF REPLICATION The single circular chromosome of the bacterium E. coli has 4 × 10⁶ base pairs (bp) of DNA. The 245 bp ori sequence is at a particular location on the chromosome. Once the pre-replication complex binds to it, the DNA is unwound and replication proceeds in both directions around the circle, forming two replication forks (Figure 13.10A). The replication rate in E. coli is approximately 1,000 bp per second, so it takes about 40 minutes to fully replicate the chromosome (with two replication forks). Rapidly dividing E. coli cells divide every 20 minutes. In these cells, new rounds of replication begin at the ori of each new chromosome before the first chromosome has fully replicated. In this way the cells can divide more frequently than the time needed to finish replicating the original chromosome.

Figure 13.10 The Origin of DNA Replication (A) Prokaryotic chromosomes usually have a single origin where DNA replication starts and proceeds in both directions. (B) The usually much larger eukaryotic chromosome typically has multiple replication origins.

Eukaryotic chromosomes are typically much longer than those of prokaryotes—up to 1 billion bp—and are linear, not circular. If replication occurred from a single ori with two forks growing away from each other, it would take weeks to fully replicate a chromosome. So eukaryotic chromosomes have multiple origins of replication, scattered at intervals of 10,000 to 40,000 bp (Figure 13.10B).

DNA REPLICATION BEGINS WITH A PRIMER A DNA polymerase elongates a polynucleotide strand by covalently linking new nucleotides to a preexisting strand. However, it cannot start this process without a short “starter” strand, called a primer. In most organisms this primer is a short single strand of RNA (Figure 13.11), but in some organisms it is DNA. The primer is complementary to the DNA template and is synthesized one nucleotide at a time by an enzyme called a primase. The DNA polymerase then adds nucleotides to the 3′ end of the primer and continues until the replication of that section of DNA has been completed. Then the RNA primer is degraded, DNA is added in its place, and the resulting DNA fragments are connected by the action of other enzymes. When DNA replication is complete, each new strand consists only of DNA.

Figure 13.11 DNA Forms with a Primer DNA polymerases require a primer—a “starter” strand of DNA or RNA to which they can add new nucleotides.

DNA POLYMERASES ARE LARGE DNA polymerases are much larger than their substrates (the dNTPs) and the template DNA, which is very thin. Molecular models of the enzyme–substrate–template complex from bacteria show that the enzyme is shaped like an open right hand with a palm, a thumb, and fingers (Figure 13.12). Within the “palm” is the active site of the enzyme, which brings together each dNTP substrate and the template. The “finger” regions have precise shapes that can recognize the different shapes of the four nucleotide bases. They bind to the bases by hydrogen bonding and rotate inward. Most cells contain more than one kind of DNA polymerase, but only one of them is responsible for chromosomal DNA replication. The others are involved in primer removal and DNA repair. Fifteen DNA polymerases have been identified in humans, whereas the bacterium E. coli has five DNA polymerases.

Figure 13.12 DNA Polymerase Binds to the Template Strand (A) The DNA polymerase enzyme (tan) is much larger than the DNA molecule (red and blue).(B) DNA polymerase is shaped like a hand, and in this side-on view, its “fingers” can be seen curling around the DNA. These “fingers” can recognize the distinctive shapes of the four bases.

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