Property	Prokaryotes	Eukaryotes
Typical size	0.4–4 μm	5–50 μm
Nucleus	−	+
Membrane-bounded organelles	−	+
Cytoskeleton	−	+
Endocytosis and exocytosis	−	+
Cell wall	+ (some −)	+ (plants)
		− (animals)
No. of chromosomes	1 (+plasmids)	>1
Ploidy	Haploid	Haploid or diploid
Histones	−	+
Introns	−	+
Ribosomes	70S	80S

This chapter describes DNA replication and protein synthesis in prokaryotes. The corresponding processes in eukaryotes (see Chapter 7) are similar but are often more complex.

DNA contains four bases

DNA is a polymer of nucleoside monophosphates (also called nucleotides) (Fig. 6.3, B). Its structural backbone consists of alternating phosphate and 2-deoxyribose residues that are held together by phosphodiester bonds involving carbon-3 and carbon-5 of the sugar. Carbon-1 forms a β-N-glycosidic bond with one of the four bases shown in Figure 6.4.

Figure 6.3 The building blocks of DNA. A, Structures of the four bases, 2-deoxyribose, and phosphate. The bases A and G are purines, and C and T are pyrimidines. B, Structure of 2-deoxy-adenosine monophosphate (dAMP), one of the four 2-deoxyribonucleoside monophosphates (also called 2-deoxynucleotides) in the repeat structure of DNA. Note that a nitrogen atom of the base is bound by a β-N-glycosidic bond to C-1 of 2-deoxyribose, whereas C-5 forms a phosphate ester bond.

Figure 6.4 Structure of the (2-deoxy-)tetranucleotide ACTG. The DNA strands in chromosomes are far larger, with lengths of many million nucleotide units. A, Adenine; C, cytosine; G, guanine; T, thymine.

One end of the DNA strand has a free hydroxyl group at C-5 of the last 2-deoxyribose. The other end has a free hydroxyl group at C-3. The carbons of 2-deoxyribose are numbered by a prime (′) to distinguish them from the carbon and nitrogen atoms of the bases; therefore, each strand has a 5′ end and a 3′ end. By convention, the 5′ terminus of a DNA (or RNA) strand is written at the left end and the 3′ terminus at the right end. Thus the tetranucleotide in Figure 6.4 can be written as ACTG but not GTCA.

The variability of DNA structure is produced by its base sequence. With four different bases, there are 4² (or 16) different dinucleotides and 4³ (or 64) different trinucleotides, and 4¹⁰⁰ possibilities exist for a sequence of 100 nucleotides.

DNA forms a double helix

Cellular DNA is double stranded, and almost all of it is present as a double helix, as first described by James Watson and Francis Crick in 1953. The most prominent features of the Watson-Crick double helix (Figs. 6.5 to 6.7) are as follows:

1. The two strands of the double helix have opposite polarity, meaning they run in opposite directions. Base pairing is always antiparallel, not only in the DNA double helix but also in other base-paired structures formed by DNA or RNA.

2. The 2-deoxyribose/phosphate backbones of the two strands form two ridges on the surface of the molecule. The phosphate groups are negatively charged.

3. The bases face inward to the helix axis, but their edges are exposed. They form the lining of two grooves that are framed by the ridges of the sugar-phosphate backbone. Because the N-glycosidic bonds are not exactly opposite each other (see Fig. 6.7), the two grooves are of unequal size. They are called the major groove and the minor groove.

4. In each of the two strands, successive bases lie flat, one on top of the other, like a stack of pancakes. The flat surfaces of the bases are hydrophobic, and successive bases in a strand form numerous van der Waals interactions.

5. Bases in opposite strands interact by hydrogen bonds. Adenine (A) always pairs with thymine (T) in the opposite strand, and guanine (G) with cytosine (C). Therefore the molar amount of adenine in the double-stranded DNA always equals that of thymine, and the amount of guanine equals that of cytosine. A-T base pairs are held together by two hydrogen bonds, and G-C base pairs by three. Most important, the base sequence of one strand predicts exactly the base sequence of the opposite strand. This is essential for DNA replication and DNA repair.

6. The double strand is wound into a right-handed helix. Each turn of the helix has about 10.4 base pairs and advances about 3.4 nm along the helix axis. The double helix is rather stiff, but it can be bent and twisted by DNA-binding proteins.

Figure 6.5 Schematic view of the DNA double strand. Note that the strands are antiparallel and that only A-T and G-C base pairs are permitted. Therefore the base sequence of one strand predicts the base sequence of the opposite strand. A, Adenine; C, cytosine; G, guanine; P, phosphate; T, thymine.

Figure 6.6 Space-filling model of the Watson-Crick double helix (B-DNA).

Figure 6.7 Cross-sections through an adenine-thymine (A-T) and a guanine-cytosine (G-C) base pair in the DNA duplex. The A-T base pair is held together by two hydrogen bonds [] and the G-C base pair by three.

DNA can be denatured

Like other noncovalent structures, the Watson-Crick double helix disintegrates at high temperatures. Heat denaturation of DNA is also called melting. Because A-T base pairs are held together by two hydrogen bonds and G-C base pairs by three, A-T–rich sections of the DNA unravel more easily than G-C–rich regions when the temperature is raised (Figs. 6.8 and 6.9). At physiological pH and ionic strength, this typically happens between 85°C and 95°C.

Figure 6.8 Melting of DNA.

Figure 6.9 Melting of DNA, monitored by the increase of ultraviolet light absorbance at 260 nm. The melting temperature increases with increased guanine-cytosine (G-C) content of the DNA. It is also affected by ionic strength and pH. The melting temperature is the temperature at which the increase in ultraviolet absorbance is half-maximal.

Heat denaturation decreases the viscosity of DNA solutions because the single strands are more flexible than the stiff, resilient double helix. It also increases the ultraviolet light absorbance at 260 nm, which is caused by the bases, because base pairing and base stacking are disrupted.

Other ways to denature DNA include decreased salt concentration, extreme pH, and chemicals that disrupt hydrogen bonding or base stacking.

When cooled slowly, denatured DNA “renatures” spontaneously. This process is called annealing. Whereas small DNA molecules anneal almost instantaneously, large molecules require seconds to minutes.

DNA is supercoiled

Many naturally occurring DNA molecules are circular. When a linear duplex is partially unwound by one or several turns before it is linked into a circle, the number of base pairs per turn of the helix is greater than the usual 10.4. The torsional strain in this molecule leads to supercoiling of the duplex around its own axis, much as a telephone cord twists around itself. This is called a negative supertwist. The opposite situation, in which the helix is overwound, is called a positive supertwist.

Most cellular DNAs are negatively supertwisted, with 5% to 7% fewer right-handed turns than expected from the number of their base pairs. This underwound condition favors the unwinding of the double helix during DNA replication and transcription.

The supertwisting of DNA is regulated by two types of topoisomerase. Type I topoisomerases cleave one strand of the double helix, creating a molecular swivel that relaxes supertwists passively. Type II topoisomerases are more complex. They cleave both strands and allow an intact helix to pass through this transient double-strand break, before resealing the break. Type II topoisomerases hydrolyze ATP to pump negative supertwists into the DNA (Fig. 6.10).

Figure 6.10 Relaxation of positive supertwists in DNA by a type II topoisomerase.

DNA replication is semiconservative

DNA makes identical copies of itself, which are transmitted to the daughter cells during mitosis and even to the next generation through the gametes. In this sense, DNA is the only immortal molecule in the body. The organism is best understood as an artificial environment, created by genes for the benefit of their own continued existence.

DNA is replicated in two steps (Fig. 6.11):

1. The double helix unwinds to produce two single strands. This requires ATP-dependent enzymes to break the hydrogen bonds between bases. DNA unwinding creates the replication fork. This is the place where the new DNA strands are synthesized.

2. A new complementary strand is synthesized for each of the two old strands. This is possible because the base sequence of each strand predicts the base sequence of the complementary strand.

Figure 6.11 Semiconservative mechanism of DNA replication.

DNA replication is called semiconservative because one strand in the daughter molecule is always old and the other strand is newly synthesized.

DNA is synthesized by DNA polymerases

The steps in DNA replication are best known in Escherichia coli, an intestinal bacterium that has enjoyed the unfaltering affection of generations of molecular biologists.

The key enzymes of DNA replication in E. coli, as in all other cells, are the DNA polymerases. DNA polymerases synthesize the new DNA strand stepwise, nucleotide by nucleotide, in the 5′→3′ direction. The precursors are the deoxyribonucleoside triphosphates: deoxy-adenosine triphosphate (dATP), deoxy-guanosine triphosphate (dGTP), deoxy-cytosine triphosphate (dCTP), and deoxy-thymidine triphosphate (dTTP). DNA polymerase elongates DNA strands by linking the proximal phosphate of an incoming nucleotide to the 3′-hydroxyl group at the end of the growing strand (Fig. 6.12). The pyrophosphate formed in this reaction is rapidly cleaved to inorganic phosphate by cellular pyrophosphatases.

Figure 6.12 Template-directed synthesis of DNA by DNA polymerases. A, Adenine; C, cytosine; G, guanine; P, phosphate; T, thymine.

Prior unwinding of the double helix is required because the DNA polymerases require a single-stranded DNA as a template. While synthesizing the new strand in the 5′→3′ direction, the enzyme moves along the template strand in the 3′→5′ direction.

DNA polymerases are literate enzymes. They read the base sequence of their template and make sure that each base that they add to the new strand pairs with the base in the template strand. Therefore the new strand is exactly complementary to the template strand. The DNA polymerases are lacking in creative spirit. They are like the scribe monks in medieval monasteries, who worked day and night copying old manuscripts without understanding their content.

Bacterial DNA polymerases have exonuclease activities

A nuclease is an enzyme that cleaves phosphodiester bonds in a nucleic acid. Deoxyribonucleases (DNases) cleave DNA, and ribonucleases (RNases) cleave RNA. Nucleases that cleave internal phosphodiester bonds are called endonucleases, and those that cleave bonds at the 5′ end or the 3′ end are called exonucleases.

Nobody is perfect, and even DNA polymerase sometimes incorporates a wrong nucleotide in the new strand. This can create a lasting mutation, which can be deadly if it leads to the synthesis of a faulty protein. To minimize such mishaps, the bacterial DNA polymerases are equipped with a 3′-exonuclease activity that they use for proofreading. When the nucleotide that has been added to the 3′ end of a growing chain fails to pair with the base in the template strand, it is removed by the 3′-exonuclease activity (Fig. 6.13). This proofreading mechanism reduces the error rate from 1 in 10⁴ or 1 in 10⁵ to less than 1 in 10⁷.

Figure 6.13 Exonuclease activities of bacterial DNA polymerases. The products of these cleavages are nucleoside 5′-monophosphates. A, 3′-Exonuclease activity. Only mismatched bases are removed from the 3′ end of the newly synthesized DNA strand. This activity is required for proofreading. B, 5′-Exonuclease activity. Base-paired nucleotides are removed from the 5′ end. This activity is required to erase the RNA primer during DNA replication and to remove damaged portions of DNA during DNA repair.

Most bacterial DNA polymerases also have a 5′-exonuclease activity (see Fig. 6.13). This activity is not used for proofreading, but it cleaves damaged DNA strands during DNA repair and erases the RNA primer during DNA replication.

Escherichia coli has three DNA polymerases. They differ in their affinity for the DNA template and consequently in their processivity, the number of nucleotides they polymerize before dissociating from the template:

• Poly I has low processivity and falls off its template after polymerizing only a few dozen nucleotides. This enzyme is used for DNA repair (see Chapter 9) and plays only an accessory role in DNA replication.

• Poly II has a somewhat higher processivity. It is involved with DNA repair as well.

• Poly III is the major enzyme of DNA replication. With the help of a specialized clamp protein, it binds tightly to its template and polymerizes hundreds of thousands of nucleotides in one sitting, at a rate of about 800 nucleotides per second.