BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 24 DNA: THE GENETIC ROLE, STRUCTURE, AND REPLICATION

24.10. Some DNA Molecules Are Circular

Electron Cell/15.html">Microscopy has revealed that intact DNA molecules from many sources are closed into rings (Fig. 24.18). The discovery that E. coli has a circular chromosome came as no surprise. It had been predicted on The basis of genetic studies showing that the genetic linkage map of this bacterium is circular. The term "circular" simply means that the DNA chain is continuous and does not refer to its geometric shape. In vivo, DNA molecules always assume highly compact forms. In this regard, it is noteworthy that the E. coli chromosome is approximately 1,000 times longer than the bacterial cell itself.

Not all DNA molecules are circular. For example, the DNA of bacteriophage T7 is linear. The DNA molecules of certain Viruses, such as bacteriophage , undergo interconversion between linear and circular forms. The linear form is present in the viral particle, whereas the circular form is found in the host cell (Section 30.16).

24.11. Circular Duplex DNA Molecules Can Exist in a Supercoiled State

When a linear double-stranded DNA molecule is converted into a closed circular molecule, it acquires a novel property. Jerome Vinograd discovered that the axis of the DNA double helix can itself be twisted into a superhelix. This superhelix can be right-handed or left-handed (Fig. 24.22). The terms "supercoiled," "superhelical," and "supertwisted" are synonymous. Circular DNA completely devoid of superhelical turns is called relaxed. Converting relaxed DNA into a supercoiled form requires a specific input of energy. For instance, the energy required to introduce 15 superhelical turns into a single SV-40 virus DNA molecule (with a contour length of 1.7 µm) is about 100 kcal/mol. The strain energy of supercoiled DNA (supercoiling energy) is approximately proportional to the square of the number of superhelical turns.

Class="center">Fig. 24.22. Schematic representation of relaxed (A) and supercoiled (B) DNA

Supercoiling apparently serves two biological Functions. First, supercoiled DNA is more compact than relaxed DNA of the same length (Fig. 24.23). Supercoiling may play a role in DNA packaging. Second, supercoiling can influence the degree of unwinding of The Double Helix and, consequently, its interactions with other molecules. Specifically, negative supercoiling can promote the unwinding of the double helix. It is interesting to note that almost all naturally occurring circular DNA molecules are negatively supercoiled.

Fig. 24.23. Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF Mitochondrial DNA: A — relaxed circular form; B — supercoiled circular form

An important characteristic of closed Circular DNA is its linking number, L. The number L indicates how many times one strand crosses the other when projected onto a plane. The number L must be an integer. The twist, T, and the writhe (a measure of supercoiling), W, are related by the equation

L = W + T, i.e., they are inversely related.

The linking number is a topological property; it can change only if breaks are introduced into one or both strands of the circular DNA. Indeed, Enzymes that catalytically alter the value of L have been isolated. The catalytic activity of such topoisomerases is readily detected by gel Electrophoresis, because supercoiled DNA is more compact and therefore migrates faster than relaxed DNA (Fig. 24.24).

Fig. 24.24. Gel photographs showing the relaxation of SV-40 virus supercoiled DNA. Lane A — supercoiled DNA with a high number of negative turns. Upon incubation of the DNA with topoisomerase for 5 min (B) and 30 min (C), a series of bands with lower degrees of supercoiling is generated

24.12. Discovery of DNA Polymerase

We now turn to the Molecular Mechanism of METABOLISM/36.html">DNA Replication. In 1955, Arthur Kornberg and his colleagues began searching for an enzyme that synthesizes DNA. This search was soon crowned with success, largely because three sound decisions were made during the experimental design—that is, correct choices were made among several possibilities.

1. What are the activated precursors of DNA? Kornberg and his colleagues correctly surmised that the activated intermediates in DNA Synthesis are deoxynucleoside 5'-triphosphates. This hypothesis was based on two considerations. First, the biosynthetic pathways of Purines and Pyrimidines lead to The formation of nucleoside 5'-phosphates (rather than nucleoside 3'-phosphates). Second, the activated intermediate in the synthesis of pyrophosphate bonds in Coenzymes such as NAD+, FAD, and CoA is ATP.

2. What can serve as a criterion for DNA synthesis? It was anticipated that the absolute amount of DNA synthesized in the initial experiments would be very small, largely due to the Abundance of Nucleases. Therefore, a sensitive assay was required. Such an assay was developed using radioactive nucleotide precursors.

The incorporation of these precursors into DNA was detected by measuring the radioactivity in the precipitate obtained after treating the incubation mixture with acid. This method relies on the fact that DNA is precipitated by acids, such as trichloroacetic acid, whereas precursor NUCLEOTIDES remain in solution.

3. Which Cells should be chosen for study? After initial experiments with animal cell extracts yielded negative results, E. coli Bacteria were selected because these cells divide in a mere 20 minutes (generation time) and can therefore be grown in large quantities. As expected, this bacterium is exceptionally rich in enzymes involved in DNA synthesis.

The E. coli extract was incubated with radioactive deoxythymidine-5'-triphosphate. The radioactivity of this ¹⁴C-labeled precursor was 1 • 10⁶ cpm. The radioactivity of the precipitate obtained by adding acid to the incubation mixture was only 50 cpm. Although only a few picomoles of DNA were synthesized, this marked the beginning. Kornberg wrote: “Although The amount of nucleotide incorporated into the nucleic acid was negligible, it was significantly above Background. We tried to drive a wedge into this tiny crack. Our hammer was Enzyme Purification — a method refined through The Study of Alcoholic Fermentation.”

This new enzyme was named DNA polymerase (Fig. 24.25). It is now referred to as DNA polymerase I, as other DNA polymerases have since been isolated. After a decade of intensive work in Kornberg’s laboratory, DNA polymerase I was purified to homogeneity and characterized in detail. The sheer scale of the effort is reflected in the fact that obtaining 500 mg of pure enzyme required starting with 100 kg of E. coli cells.

Fig. 24.25. Electron micrograph of DNA polymerase molecules (spherical structures) bound to a DNA molecule (thin filament)

DNA polymerase I is a single polypeptide chain with a mass of 109 kDa. It catalyzes the sequential addition of deoxyribonucleotide units to a DNA chain:

For DNA chain synthesis, DNA polymerase I requires the following components:

1. All four deoxyribonucleoside-5'-triphosphates — dATP, dGTP, dTTP, and dCTP — must be present in the medium. Hereinafter, we will refer to these deoxyribonucleoside triphosphates collectively as dNTP. In addition, Mg²⁺ ions are required.

2. DNA polymerase I adds deoxyribonucleotides to the 3'-hydroxyl terminus of a preexisting DNA (or RNA) strand. In other words, a primer strand, or primer, with a free 3'-hydroxyl group is required.

3. A DNA template is required. Both single- and double-stranded DNA can serve as templates. Double-stranded DNA functions as an effective template only if its sugar-phosphate backbone is nicked in one or more places.

The chain elongation reaction catalyzed by DNA polymerase proceeds via a nucleophilic attack by the 3'-OH end of the template on the phosphorus atom closest to the ribose of the incoming deoxyribonucleoside triphosphate. This results in the formation of a phosphodiester bridge with the simultaneous release of pyrophosphate (Fig. 24.26). Subsequent Hydrolysis of the pyrophosphate drives further polymerization. Such a shift in the overall equilibrium would be impossible if nucleoside diphosphates served as the activated intermediates. This is precisely why we see a compelling reason for the predominance of nucleoside triphosphates over diphosphates as activated precursors in biosynthetic reactions. DNA chain elongation proceeds in the 5' → 3' direction (Fig. 24.27). A single molecule of DNA polymerase I incorporates approximately 10 nucleotides per second. The polymerization is processive, meaning that the enzyme adds many nucleotides while remaining bound to a single template.

Fig. 24.26. Chain elongation reaction catalyzed by DNA polymerase

Fig. 24.27. DNA polymerases catalyze the growth of DNA chains in the 5' → 3' direction