Chapter 10: Genetic Material & DNA - BIO3500 PDF

## 10.1 Genetic Material Possesses Several Key Characteristics Life is characterized by tremendous diversity, but the coding instructions for all living organisms are written in the same genetic language - that of nucleic acids. Surprisingly, the idea that genes are made of nucleic acids was not wi...

## 10.1 Genetic Material Possesses Several Key Characteristics Life is characterized by tremendous diversity, but the coding instructions for all living organisms are written in the same genetic language - that of nucleic acids. Surprisingly, the idea that genes are made of nucleic acids was not widely accepted until after 1950. This skepticism was due in part to a lack of knowledge about the structure of deoxyribonucleic acid (DNA). Until the structure of DNA was understood, no one knew how DNA could store and transmit genetic information. Even before nucleic acids were identified as the genetic material, biologists recognized that whatever the nature of the genetic material, it must possess four important characteristics: 1. The genetic material must contain complex information. First and foremost, the genetic material must be capable of storing large amounts of information - instructions for the traits and functions of an organism. 2. The genetic material must replicate faithfully. Every organism begins life as a single cell. To produce a complex multicellular organism like yourself, this single cell must undergo billions of cell divisions. At each cell division, the genetic instructions must be accurately transmitted to descendant cells. And when organisms reproduce and pass genes to their progeny, the genetic instructions must be copied with fidelity. 3. The genetic material must encode the phenotype. The genetic material (the genotype) must have the capacity to be expressed as a phenotype - to code for traits. The product of a gene is often a protein or an RNA molecule, so there must be a mechanism for genetic instructions in the DNA to be copied into RNAs and proteins. 4. The genetic material must have the capacity to vary. Genetic information must have the ability to vary, because different species - and even individual members of the same species - differ in their genetic makeup. ## Early Studies of DNA In 1868, Johann Friedrich Miescher (Figure 10.2) graduated from medical school in Switzerland. Influenced by an uncle who believed that the key to understanding disease lay in the chemistry of tissues, Miescher traveled to Tübingen, Germany, to study under Ernst Felix Hoppe-Seyler, an early leader in the emerging field of biochemistry. Under Hoppe-Seyler's direction, Miescher turned his attention to the chemistry of pus, a substance of clear medical importance. Pus contains white blood cells, which have large nuclei, and Miescher developed a method for isolating these nuclei. The minute amounts of nuclear material that he obtained were insufficient for a thorough chemical analysis, but he did establish that the nuclear material contained a novel substance that was slightly acidic and high in phosphorus. This material, which we now know must have consisted of DNA and protein, Miescher called nuclein. The substance was later renamed nucleic acid by one of his students. **Image Description (Figure 10.2)** (a) A portrait of a man with a receding hairline and a mustache, wearing a suit. (b) An old, black and white photo of a room with a table and chairs, with old scientific equipment, such as glassware and beakers. By 1887, several researchers had independently concluded that the physical basis of heredity lies in the nucleus. Chromatin was shown to consist of nucleic acid and proteins, but which of these substances was actually the genetic information was not clear. In the late 1800s, Albrecht Kossel carried out further work on the chemistry of DNA and determined that it contains four nitrogenous bases: adenine, cytosine, guanine, and thymine (abbreviated A, C, G, and T). In the early twentieth century, the Rockefeller Institute in New York City became a center for nucleic acid research. Phoebus Aaron Levene joined the Institute in 1905 and spent the next 40 years studying the chemistry of DNA. He discovered that DNA consists of a large number of linked, repeating units, called nucleotides; each nucleotide contains a sugar, a phosphate, and a base. ## Nucleotide Levene incorrectly proposed that DNA consists of a series of four-nucleotide units, each containing all four bases - adenine, guanine, cytosine, and thymine - in a fixed sequence. This concept, known as the tetranucleotide hypothesis, implied that the structure of DNA was not variable enough to make it the genetic material. The tetranucleotide hypothesis contributed to the idea that protein is the genetic material because the structure of protein, with its 20 different amino acids, could be highly variable. As additional studies of the chemistry of DNA were completed in the 1940s and 1950s, the notion of DNA as a simple, invariant molecule began to change. Erwin Chargaff and his colleagues carefully measured the amounts of the four bases in DNA from a variety of organisms, and they found that DNA from different organisms varies greatly in base composition. This finding disproved the tetranucleotide hypothesis. They discovered that within each species, there is some regularity in the ratios of the bases: the amount of adenine is always equal to the amount of thymine (A = T), and the amount of guanine is always equal to the amount of cytosine (G = C) (Table 10.1). These findings became known as Chargaff's rules. However, the cause of these ratios among the bases was unknown at the time. ## DNA as the Source of Genetic Information While chemists were working out the structure of DNA, biologists were attempting to identify the carrier of genetic information. Mendel identified the basic rules of heredity in 1866, but he had no idea about the physical nature of hereditary information. By the early 1900s, biologists had concluded that genes resided on chromosomes, which were known to contain both DNA and protein. Two sets of experiments, one conducted on bacteria and the other on viruses, provided pivotal evidence that DNA, rather than protein, was the genetic material. ## The Discovery of the Transforming Principle An initial step in identifying DNA as the source of genetic information came with the discovery of a phenomenon called transformation (described in Section 9.3). This phenomenon was first observed in 1928 by Fred Griffith, an English physician whose special interest was the bacterium that causes pneumonia: Streptococcus pneumoniae. Griffith had succeeded in isolating several different strains of S. pneumoniae (type I, II, III, and so forth). In the virulent (disease-causing) forms of a strain, each bacterium is surrounded by a polysaccharide coat, which makes the bacterial colony appear smooth (S) when grown on an agar plate. Griffith found that these virulent forms occasionally mutated to nonvirulent forms, which lack a polysaccharide coat and produce a rough-appearing colony (R). Griffith observed that small amounts of living type IIIS bacteria injected into mice caused the mice to develop pneumonia and die; when he examined the dead mice, he found large amounts of type IIIS bacteria in their blood (Figure 10.3a). When Griffith injected type IIR bacteria into mice, the mice lived, and no bacteria were recovered from their blood (Figure 10.3b). Griffith knew that boiling killed all bacteria and destroyed their virulence; when he injected large amounts of heat-killed type IIIS bacteria into mice, the mice lived, and no type IIIS bacteria were recovered from their blood (Figure 10.3c). **Image Description (Figure 10.3)** A diagram with four separate illustrations showing vials of bacteria being injected into a mouse. (a) Vial labeled "Type IIIS (virulent) bacteria are injected into a mouse". Result: "Mouse dies". (b) Vial labeled "Type IIR (nonvirulent) bacteria are injected into a mouse". Result: "Mouse lives". (c) Vial labeled "Heat-killed type IIIS bacteria are injected into a mouse". Result: "Mouse lives". (d) Vial labeled "A mixture of type IIR bacteria and heat-killed type IIIS bacteria is injected into a mouse". Result: "Mouse dies". Griffith observed that small amounts of living type IIIS bacteria injected into mice caused the mice to develop pneumonia and die; when he examined the dead mice, he found large amounts of type IIIS bacteria in their blood (Figure 10.3a). When Griffith injected type IIR bacteria into mice, the mice lived, and no bacteria were recovered from their blood (Figure 10.3b). Griffith knew that boiling killed all bacteria and destroyed their virulence; when he injected large amounts of heat-killed type IIIS bacteria into mice, the mice lived, and no type IIIS bacteria were recovered from their blood (Figure 10.3c). **Image Description (Figure 10.3)** A diagram with four separate illustrations showing vials of bacteria being injected into a mouse. (a) Vial labeled "Type IIIS (virulent) bacteria are injected into a mouse". Result: "Mouse dies". (b) Vial labeled "Type IIR (nonvirulent) bacteria are injected into a mouse". Result: "Mouse lives". (c) Vial labeled "Heat-killed type IIIS bacteria are injected into a mouse". Result: "Mouse lives". (d) Vial labeled "A mixture of type IIR bacteria and heat-killed type IIIS bacteria is injected into a mouse". Result: "Mouse dies". ## Identification of the Transforming Principle At the time of Griffith's report, Oswald Avery was a microbiologist at the Rockefeller Institute. At first, Avery was skeptical of Griffith's results, but after other microbiologists successfully repeated Griffith's experiments with other bacteria, Avery set out to understand the nature of the transforming substance. After 10 years of research, Avery, Colin MacLeod, and Maclyn McCarty succeeded in isolating and partially purifying the transforming substance. They showed that it had a chemical composition closely matching that of DNA and quite different from that of proteins. Enzymes such as trypsin and chymotrypsin, known to break down proteins, had no effect on the transforming substance. Ribonuclease, an enzyme that destroys RNA, also had no effect. Enzymes capable of destroying DNA, however, eliminated the biological activity of the transforming substance (Figure 10.4). Avery, MacLeod, and McCarty showed that the transforming substance precipitated at about the same rate as purified DNA and that it absorbed ultraviolet light at the same wavelengths as DNA. These results, published in 1944, provided compelling evidence that the transforming principle - and therefore genetic information - resides in DNA. However, new theories in science are rarely accepted on the basis of a single experiment, and many biologists continued to prefer the hypothesis that the genetic material is protein. **Image Description (Figure 10.4)** A diagram with five illustrations showing vials of bacteria and associated steps involved in an experiment. 1. Vial labeled "Type IIIS (virulent) bacteria". Step: "Use heat to kill virulent bacteria, homogenize, and filter". 2. Vial labeled "Type IIIS bacterial filtrate". Step: "Treat samples with enzymes that destroy proteins, RNA, or DNA". 3. Vial labeled "Protease (destroys proteins)". Vial labeled "RNase (destroys RNA)". Vial labeled "DNase (destroys DNA)". Step: "Add the treated samples to cultures to type IIR bacteria". 4. Vial labeled "Type IIR bacteria". Vial labeled "Type IIR bacteria". Vial labeled "Type IIR bacteria". Step: "Cultures treated with protease or RNase contain transformed type IIIS bacteria,...". 5. Vial labeled "Type IIR bacteria". Step: "...but the culture treated with DNase does not." Conclusion: "Because only DNase destroyed the transforming substance, the transforming substance is DNA." ## The Hershey-Chase Experiment A second piece of evidence that indicated DNA was the genetic material resulted from a study of the T2 bacteriophage conducted by Alfred Hershey and Martha Chase (Figure 10.5a). The T2 bacteriophage is a virus that infects the bacterium Escherichia coli. As we saw in Section 9.5, a phage reproduces by attaching to the outer wall of a bacterial cell and injecting its DNA into the cell, where it replicates and directs the cell to synthesize phage proteins. The phage DNA becomes encapsulated within the phage proteins, producing progeny phages that lyse (break open) the cell and escape (Figure 10.5b). **Image Description (Figure 10.5)** (a) An illustration showing a T2 bacteriophage attached to an E. coli bacterium, drawn to scale. The legend states that the "phage genome is DNA" and "all other parts of the bacteriophage are protein". (b) A diagram called "The life cycle of the T2 bacteriophage". A phage attaches to an E. coli bacterium and injects its chromosome. The E. coli's chromosome breaks down, and the phage chromosome replicates. Expression of phage genes produces phage structural components. Progeny phages assemble, and the bacterial wall lyses, releasing progeny phages. At the time of the Hershey-Chase study (their paper was published in 1952), biologists did not understand exactly how phages reproduce. What they did know was that the T2 phage is approximately 50% protein and 50% DNA, that a phage infects a bacterial cell by first attaching to the cell wall, and that progeny phages are ultimately produced within the cell. Because the progeny carry the same traits as the infecting phage, genetic material from the infecting phage must be transmitted to the progeny, but how this genetic transmission takes place was unknown. Hershey and Chase designed a series of experiments to determine whether the phage protein or the phage DNA is transmitted in phage reproduction. To follow the fates of protein and DNA, they used radioactive forms, or isotopes, of phosphorus and sulfur. A radioactive isotope can be used as a tracer to identify the location of a specific molecule, because any molecule containing the isotope will be radioactive and therefore easily detected. DNA contains phosphorus but not sulfur, so Hershey and Chase used a radioactive isotope of phosphorus (32P) to follow phage DNA during reproduction. Protein contains sulfur but not phosphorus, so they used a radioactive isotope of sulfur (35S) to follow the protein. Hershey and Chase grew one batch of E. coli in a medium containing 32P and infected the bacteria with the T2 phage so that all the progeny phages would have DNA labeled with 32P (Figure 10.6). They grew a second batch of E. coli in a medium containing 35S and infected these bacteria with T2 phage so that all the progeny phages would have proteins labeled with 35S. Hershey and Chase then infected separate batches of unlabeled E. coli with the 35S- and 32P-labeled progeny phages. After allowing time for the phages to infect the E. coli cells, they placed the cells in a blender and sheared off the now-empty phage protein coats from the cell walls. They separated out the protein coats and cultured the infected bacterial cells. **Image Description (Figure 10.6)** A diagram illustrating the Hershey-Chase experiment. On one side, a T2 phage is infected with "35S" and injected into "E. coli" grown in a medium containing "35S". Result: "35S" is taken up in phage protein and the progeny phages with "35S" infect unlabeled "E. coli". After centrifugation, "35S" is recovered in the supernatant. On the other side, a T2 phage is infected with "32P" and injected into "E. coli" grown in a medium containing "32P". Result: "32P" is taken up in phage DNA and the progeny phages with "32P" infect unlabeled "E. coli". After centrifugation, "32P" is recovered in the pellet at the bottom of the tube. After centrifugation, "35S" is recovered in the fluid containing the virus coats. After centrifugation, infected bacteria form a pellet containing "32P" in the bottom of the tube. Progeny phages are radioactive. Conclusion: "DNA - not protein - is the genetic material in bacteriophages." In the case of the bacteria infected by phages labeled with 35S, most of the radioactivity was detected in the phage protein coats, and little was detected in the cells. Furthermore, when new phages emerged from the cells, they contained almost no 35S (see Figure 10.6). This result indicated that the protein component of a phage does not enter the cell and is not transmitted to progeny phages. In contrast, when Hershey and Chase infected bacteria with 32P-labeled phages and then removed the phage protein coats, the bacteria were radioactive. Most significantly, after the bacterial cells were lysed and new progeny phages emerged, many of those phages emitted radioactivity, demonstrating that DNA from the infecting phages had been passed on to the progeny phages (see Figure 10.6). These results confirmed that DNA, not protein, is the genetic material of phages. ## Watson and Crick's Discovery of the Three-Dimensional Structure of DNA These experiments on the nature of the genetic material set the stage for one of the most important advances in the history of biology: the discovery of the three-dimensional structure of DNA by James Watson and Francis Crick in 1953. Before Watson and Crick's breakthrough, much of the basic chemistry of DNA had already been determined by Miescher, Kossel, Levene, Chargaff, and others, who had established that DNA consists of nucleotides and that each nucleotide contains a sugar, a nitrogenous base, and a phosphate group. However, how the nucleotides fit together in the three-dimensional structure of the molecule was not at all clear. In 1947, William Astbury began studying the three-dimensional structure of DNA by using a technique called X-ray diffraction (Figure 10.7), in which X-rays beamed at a molecule are reflected in specific patterns that reveal aspects of the structure of the molecule. However, his diffraction images did not provide enough resolution to reveal the structure. A research group at King's College in London, led by Maurice Wilkins, also used X-ray diffraction to study DNA. Working in Wilkins's laboratory, Rosalind Franklin obtained strikingly better images of the molecule. Rosalind Franklin was an excellent X-ray crystallographer, and her images were critical in the eventual elucidation of the structure of DNA by Watson and Crick. **Image Description (Figure 10.7)** An illustration showing how X-ray diffraction works. Crystals of a substance are bombarded with X-rays, which are diffracted. The spacing of the atoms within the crystal appears as spots on a photographic film. The diffraction pattern provides information about the structure of the molecule. Watson and Crick investigated the structure of DNA not by collecting new data but by using all available information about the chemistry of DNA to construct molecular models (Figure 10.8a). By using the excellent X-ray diffraction images taken by Rosalind Franklin (Figure 10.8b) and by applying the laws of structural chemistry,, they were able to limit the number of possible structures that DNA could assume. They tested various structures by building models made of wire and metal plates. With their models, they were able to see whether a structure was compatible with chemical principles and with the X-ray images. **Image Description (Figure 10.8)** (a) Photo of Watson and Crick with a model of DNA in the background. (b) An X-ray diffraction image of DNA, taken by Rosalind Franklin. The key to solving the structure came when Watson recognized that an adenine base could bond with a thymine base and that a guanine base could bond with a cytosine base; these pairings accounted for the base ratios that Chargaff had discovered earlier. The model developed by Watson and Crick showed that DNA consists of two strands of nucleotides that run in opposite directions (are antiparallel) and wind around each other to form a right-handed helix, with the sugars and phosphates on the outside and the bases in the interior. They recognized that the double-stranded structure of DNA, with its specific base pairing, provided an elegant means by which DNA could be replicated. Watson and Crick published an electrifying description of their model in Nature in 1953. At the same time, Franklin and Wilkins each published their X-ray diffraction data, which demonstrated that DNA was helical in structure. Many have called the solving of DNA's structure the most important biological discovery of the twentieth century. For their discovery, Watson and Crick, along with Maurice Wilkins, were awarded the Nobel Prize in chemistry in 1962. Rosalind Franklin had died of cancer in 1958 and thus could not be considered a candidate for the shared prize, but many scholars and historians believe that she should receive equal credit for solving the structure of DNA. Following the discovery of DNA's structure, much research was focused on how genetic information is encoded within the base sequence and how this information is copied and expressed. Even today, the details of DNA structure and function continue to be the subject of active research. ## RNA as Genetic Material In most organisms, DNA carries the genetic information. A few viruses, however, use RNA, not DNA, as their genetic material. This was demonstrated in 1956 by Heinz Fraenkel-Conrat and Bea Singer, who worked with the tobacco mosaic virus (TMV), which infects and causes disease in tobacco plants (Figure 10.9). The tobacco mosaic virus possesses a single molecule of RNA surrounded by a helically arranged cylinder of protein molecules. Fraenkel-Conrat found that after separating the RNA and the protein of TMV, he could remix the RNA and protein of different strains of TMV and obtain intact, infectious viral particles. **Image Description (Figure 10.9)** An electron micrograph showing the tobacco mosaic virus (TMV). With Singer, Fraenkel-Conrat then created hybrid viruses by mixing RNA and protein from different strains of TMV. When these hybrid viruses infected tobacco leaves, new viral particles were produced. The new viral progeny were identical to the strain from which the RNA had been isolated and did not exhibit the characteristics of the strain that donated the protein. These results showed that RNA carries the genetic information in TMV. Also in 1956, Alfred Gierer and Gerhard Schramm demonstrated that RNA isolated from TMV is sufficient to infect tobacco plants and direct the production of new TMV particles. This finding confirmed that RNA carries the genetic instructions in this virus. ## 10.3 DNA Consists of Two Complementary and Antiparallel Nucleotide Strands That Form a Double Helix DNA, though relatively simple in structure, has an elegance and beauty unsurpassed by other large molecules. It is useful to consider the structure of DNA at three levels of increasing complexity, known as the primary, secondary, and tertiary structures of DNA. The primary structure of DNA refers to its nucleotide structure and how the nucleotides are joined together. The secondary structure refers to DNA's stable three-dimensional configuration, the helical structure worked out by Watson and Crick. In Chapter 11, we will consider DNA's tertiary structures, which are the complex packing arrangements of double-stranded DNA in chromosomes. ## The Primary Structure of DNA The primary structure of DNA consists of a string of nucleotides joined together by phosphodiester linkages. ### Nucleotides DNA is typically a very long molecule and is therefore termed a macromolecule. For example, the DNA in each human chromosome is a single molecule that, if stretched out straight, would be several centimeters in length, thousands of times longer than a human cell. In spite of its large size, DNA has quite a simple structure: it is a polymer - that is, a chain made up of many repeating units linked together. The repeating units of DNA are nucleotides, each comprising three parts: (1) a sugar, (2) a phosphate group, and (3) a nitrogen-containing base. The sugars of nucleic acids - called pentose sugars - have five carbon atoms, numbered 1, 2, 3, 4, and 5 (Figure 10.10). The sugars of DNA and RNA are slightly different in structure. RNA's sugar, called ribose, has a hydroxyl group (OH) attached to the 2'-carbon atom, whereas DNA's sugar, or deoxyribose, has a hydrogen atom (H) at this position and therefore contains one oxygen atom fewer overall. This difference gives rise to the names ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). This minor chemical difference is recognized by most of the cellular enzymes that interact with DNA or RNA, thus providing specific functions for each nucleic acid. Furthermore, the additional oxygen atom in the RNA nucleotide makes it more reactive and less chemically stable than DNA. For this reason, DNA is better suited to serve as the long-term carrier of genetic information. **Image Description (Figure 10.10)** A diagram showing the structures of ribose and deoxyribose. Ribose has a hydroxyl group on the number 2 carbon, while deoxyribose has a hydrogen atom on that carbon. The second component of a nucleotide is its nitrogenous base, which may be either of two types - a purine or a pyrimidine (Figure 10.11). Each purine consists of a six-member ring attached to a five-member ring, whereas each pyrimidine consists of a six-member ring only. Both DNA and RNA contain two purines, adenine and guanine (A and G), which differ in the positions of their double bonds and in the groups attached to the six-member ring. Three pyrimidines are common in nucleic acids: cytosine (C), thymine (T), and uracil (U). Cytosine is present in both DNA and RNA; however, thymine is restricted to DNA, and uracil is found only in RNA. The three pyrimidines differ in the groups or atoms attached to the carbon atoms of the ring and in the number of double bonds in the ring. In a nucleotide, the nitrogenous base always forms a covalent bond with the 1'-carbon atom of the sugar. A deoxyribose or a ribose sugar and a base together are referred to as a nucleoside. **Image Description (Figure 10.11)** A diagram showing the structures of purine and pyrimidine bases. Adenine and guanine are purines, and cytosine, thymine, and uracil are pyrimidines. The third component of a nucleotide is its phosphate group, which consists of a phosphorus atom bonded to four oxygen atoms (Figure 10.12). Phosphate groups are found in every nucleotide and frequently carry a negative charge, which makes DNA acidic. The phosphate group is always bonded to the 5-carbon atom of the sugar (see Figure 10.10) in a nucleotide. **Image Description (Figure 10.12)** A diagram showing the structure of a phosphate group. The DNA nucleotides are properly known as deoxyribonucleotides, or deoxyribonucleoside 5-monophosphates. Because there are four types of bases, there are four different kinds of DNA nucleotides (Figure 10.13). The equivalent RNA nucleotides are termed ribonucleotides, or ribonucleoside 5'-monophosphates. RNA molecules sometimes contain additional rare bases, which are modified forms of the four common bases. These modified bases will be discussed in more detail when we examine the function of RNA molecules in Chapter 14. The names for DNA bases, nucleotides, and nucleosides are shown in Table 10.2. **Image Description (Figure 10.13)** A diagram showing the four different types of nucleotides found in DNA: deoxyadenosine 5'-monophosphate, deoxyguanosine 5'-monophosphate, deoxycytidine 5'-monophosphate, and deoxythymidine 5'-monophosphate ## Polynucleotide Strands DNA is made up of many nucleotides connected by covalent bonds, which join the 5'-phosphate group of one nucleotide to the 3'-hydroxyl group of the next nucleotide. The structures shown in Figure 10.14 are flattened into two dimensions, although the molecule itself is three-dimensional, as shown in Figure 10.15a. Note that Figure 10.14 shows only a portion of the DNA and RNA molecules; each strand continues at its '5 and 3' ends as indicated by ..... The bonds that connect nucleotides, called phosphodiester linkages, are strong covalent bonds; a series of nucleotides linked in this way constitutes a polynucleotide strand. The backbone of the polynucleotide strand is composed of alternating sugars and phosphate groups; the bases project away from the long axis of the strand. The negative charges of the phosphate groups are frequently neutralized by their association with positive charges on proteins, metals, or other molecules. **Image Description (Figure 10.14)** A diagram showing the composition of a polynucleotide strand. The strand runs in the 5'-3 direction. T-A pairs have 2 hydrogen bonds, and C-G pairs have 3 hydrogen bonds. ## Secondary Structures of DNA The secondary structure of DNA refers to its three-dimensional configuration - its fundamental helical structure. DNA's secondary structure can assume a variety of configurations, depending on its base sequence and the conditions in which it is placed. ### The Double Helix A fundamental characteristic of DNA's secondary structure is that it consists of two polynucleotide strands wound around each other - it's a double helix. The sugar-phosphate linkages are on the outside of the helix, and the bases are stacked in the interior of the molecule. The two polynucleotide strands run in opposite directions - they are antiparallel, which means that the 5' end of one strand is opposite the 3' end of the other strand. The two strands are held together by two types of molecular forces. Hydrogen bonds link the bases on opposite strands (see Figure 10.14). These bonds are relatively weak compared with the covalent phosphodiester bonds that connect the sugars and phosphate groups of adjoining nucleotides on the same strand. As we will see, several important functions of DNA require the separation of its two nucleotide strands, and this separation can be readily accomplished because of the relative ease of breaking and reestablishing the hydrogen bonds. The nature of the hydrogen bond imposes a limitation on the types of bases that can pair. Adenine normally pairs only with thymine through two hydrogen bonds, and cytosine normally pairs only with guanine through three hydrogen bonds (see Figure 10.14). Because three hydrogen bonds form between C and G and only two hydrogen bonds form between A and T, C-G pairing is stronger than A-T pairing. The specificity of the base pairing means that wherever there is an A on one strand, there must be a T in the corresponding position on the other strand, and wherever there is a G on one strand, a C must be on the other. The two polynucleotide strands of a DNA molecule are therefore not identical but rather complementary DNA strands. The complementary nature of the two nucleotide strands provides for efficient and accurate DNA replication, as we will see in Chapter 12. The second force that holds the two DNA strands together is the interaction between the stacked base pairs in the interior of the molecule. Stacking means that adjacent bases are aligned so that their rings are parallel and stack on top of one another. The stacking interactions stabilize the DNA molecule but do not require that any particular base follow another. Thus, the base sequence of the DNA molecule is free to vary, allowing DNA to carry genetic information. ## Different Secondary Structures As we have seen, DNA normally consists of two polynucleotide strands that are antiparallel and complementary (exceptions are the single-stranded DNA molecules found in a few viruses). The precise three-dimensional shape of the molecule can vary, however, depending on the conditions in which the DNA is placed and, in some cases, on the base sequence itself. The three-dimensional structure of DNA described by Watson and Crick is termed the B-DNA structure (Figure 10.15). This structure exists when plenty of water surrounds the molecule and there are no unusual base sequences in the DNA - conditions that are likely to be present in cells. The B-DNA structure is the most stable configuration for a random sequence of nucleotides under physiological conditions, and most evidence suggests that it is the predominant structure in the cell. **Image Description (Figure 10.15)** (a) A space-filling model of B-DNA, showing a right-handed helix with approximately 10 bases per turn. Minor and major grooves are visible. (b) A diagrammatic representation of B-DNA. The deoxyribose sugars are linked by phosphate. The diagram is labeled with the measurements 2nm and 3.4nm. B-DNA is a right-handed helix, meaning that it has a clockwise spiral. There are approximately 10 base pairs (bp) per 360-degree rotation of the helix, so each base pair is twisted 36 degrees relative to the adjacent bases (see Figure 10.15b). The base pairs are 0.34 nanometers (nm) apart, so each complete rotation of the molecule encompasses 3.4 nm. The diameter of the helix is 2 nm, and the bases are perpendicular to the long axis of the DNA molecule. A space-filling model shows that B-DNA has a slim and elongated structure (see Figure 10.15a). The spiraling of the nucleotide strands creates major and minor grooves in the helix, features that are important for the binding of some proteins that regulate the expression of genetic information (see Chapter 16). Another secondary structure that DNA can assume is the A-DNA structure, which exists if less water is present. Like B-DNA, A-DNA is a-right-handed helix (Figure 10.16a), but it is shorter and wider than B-DNA (Figure 10.16b), and its bases are tilted away from the main axis of the molecule. A-DNA has been detected in some DNA-protein complexes and in spores of some bacteria. **Image Description (Figure 10.16)** A diagram showing the A-DNA structure, the B-DNA structure, and the Z-DNA structure. A radically different secondary structure, called Z-DNA forms a left-handed helix. In this structure, the sugar-phosphate backbone zigzags back and forth, giving rise to its name. A Z-DNA structure can result if the molecule contains particular base sequences, such as stretches of alternating C and G nucleotides. Researchers have found that Z-DNA-specific antibodies bind to regions of the DNA that are being transcribed into RNA, suggesting that Z-DNA may play some role in gene expression. Additional secondary structures of DNA (C-DNA, D-DNA, etc.) can form under specialized laboratory conditions or in DNA with specific base sequences. ## 10.4 Special Structures Can Form in DNA and RNA Sequences within a single strand of nucleotides may be complementary to each other and able to pair by forming hydrogen bonds, producing double-stranded regions. This internal base pairing imparts a secondary structure to a single-stranded molecule. One common type of secondary structure found in single strands of nucleotides is a hairpin, which forms when sequences of nucleotides on the same strand are inverted complements. A hairpin consists of a region of paired bases (the stem) and intervening unpaired bases (a loop). When the complementary sequences are contiguous, a stem is formed with no loop (see Figure 10.18b). RNA molecules may contain numerous hairpins, which allow them to fold up into complex structures *Secondary structures play important roles in the functions of many RNA molecules, as we will see in Chapters 14 and 15.* **Image Description (Figure 10.18)** (a) A diagram called "Hairpin" showing how a single strand of DNA can fold back on itself so that complementary bases pair with each other. The stem is the double stranded portion of the helix, and the loop is the unpaired portion. (b) A diagram showing a stem without a loop. DNA sequences can also sometimes form three-stranded (triplex) structures, called H-DNA, when some of the DNA unwinds and a single polynucleotide strand from one part of the molecule pairs with double-stranded DNA from another part of the molecule (Figure 10.19). This is possible because under certain conditions, one base can simultaneously pair with two other bases. H-DNA often occurs in long sequences containing only purine bases or only pyrimidine bases. Some triplex structures consist of one strand of purines paired with two strands of pyrimidines; other triplex structures consist of one strand of pyrimidines paired with two strands of purines. Sequences capable of adopting an H-DNA conformation are common in mammalian genomes, and evidence suggests that H-DNA occurs under natural conditions. Research has demonstrated that H-DNA breaks more readily than double-stranded DNA. *Quadruplex structures involving four strands of DNA can also occur under certain conditions.* **Image Description (Figure 10.19)** A diagram showing the structure of H-DNA (triplex structure). One strand of DNA unwinds from a double helix and pairs with the double helix via hydrogen bonding. ## DNA methylation The primary structure of DNA can also be modified in various ways. One such modification is *DNA methylation*, a process in which methyl groups (CH3) are added by specific enzymes to certain positions on the nitrogenous bases. Bacterial DNA is frequently methylated to distinguish it from foreign, unmethylated DNA that may be introduced by viruses; bacteria use proteins called restriction enzymes to cut up any unmethylated viral DNA. *In eukaryotic cells, methylation is often related to gene expression. Sequences that are methylated typically show low levels of transcription, while sequences lacking methylation are actively being transcribed (see Chapter 17).* *Methylation can also affect the three-dimensional structure of the DNA molecule and is responsible for some epigenetic effects (see Chapter 21).* Adenine and cytosine are commonly methylated in bacteria. In eukaryotic DNA, cytosine bases are sometimes methylated to form 5-methylcytosine (Figure 10.20). The extent of cytosine methylation varies among eukaryotic organisms: in most animal cells, about 5% of the cytosine bases are methylated