Human Biochemistry, Volume 2 - Murray R. 1993

Structure, Function, and Replication of Informational Macromolecules
Recombinant DNA Technology
DNA

In 1944, experiments conducted by Avery, MacLeod, and McCarty demonstrated that the capacity for capsule formation in a mutant, unencapsulated pneumococcal strain could be restored by introducing purified DNA from capsule-synthesizing pneumococci into its Cells. The authors designated the DNA agent responsible for this alteration as the "transforming factor." Soon after, the transformation method became widely adopted in genetic research. More recently, experiments have been performed using Yeast cells, mammalian cells, and rodent and insect embryos as recipients, with cloned DNA serving as the donor of Genetic information.

Chemical properties of DNA

The Chemical Nature of the monomer units comprising DNA (deoxyadenylate, deoxycytidylate, deoxyguanylate, and thymidylate) is described in ch. 34. These monomers polymerize via 3', 5'-phosphodiester bonds to form a single DNA strand (Fig. 37.1). Information in DNA is encoded as a specific sequence of purine and pyrimidine deoxyribonucleotides.

As shown in the figure, the polymeric DNA molecule is polar. One end features a 5'-hydroxyl (or phosphate group), while the other terminates in a 3'-phosphate (or hydroxyl group). Based on X-Ray Diffraction data of DNA and Chargaff's rule—which states that the content of deoxyadenosine (A) residues equals that of thymidine (T), and deoxyguanosine (G) equals deoxycytidine (C) in a DNA molecule—Watson, Crick, and Wilkins proposed the double-helical model of DNA in the early 1950s. The B-form model of DNA is illustrated in Fig. 37.2. The two strands of this right-handed, double-helical molecule are held together by Hydrogen Bonds formed between purine and pyrimidine bases. Complementary base pairing is strictly specific: A always pairs with T, and G with C (Fig. 37.3).

In a double-stranded molecule, constraints imposed by restricted rotation around the phosphodiester bond, the preferential "autoconfiguration of glycosidic bonds" (Fig. 34.9), and the prevailing tautomeric forms of the four bases (A, G, T, and C, Fig. 34.3) create conditions where A can form a stable pair exclusively with T, and G exclusively with C (Fig. 37.3). This precisely accounts for Chargaff's rules (A = T; G = C). The two strands of The Double Helix, being polar, are also antiparallel; that is, one strand runs in the 5'→3' direction, while the other runs 3'→5'. This arrangement resembles two parallel one-way streets with traffic flowing in opposite directions. One of the two complementary DNA strands, containing the structural information for a specific Gene as a unique nucleotide sequence, is typically termed the coding (or template) strand, whereas the complementary strand is referred to as the noncoding strand.

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Fig. 37.1. Fragment of the DNA molecular Structure, in which the purine and pyrimidine bases adenine (A), thymine (T), cytosine (C), and guanine (G) are held together by a phosphodiester backbone connecting 2'-deoxyribosyl residues linked via N-glycosidic bonds to their respective nucleobases. Note that the phosphodiester backbone of a single DNA strand possesses "polarity" (i.e., it exhibits a defined direction, such as 5'→3').

As illustrated in Fig. 37.3, three Hydrogen bonds are formed between deoxyguanosine and deoxycytidine residues, whereas only two are formed between thymidine and deoxyadenosine. Consequently, the G—C bond is approximately 50% stronger. This circumstance, along with stacking interactions, accounts for the higher Denaturation (melting) Temperature of G—C-rich regions in DNA.

Introduction/20.html">DNA Structure

DNA can form several types of Double helices. Six forms (ranging from A to E and the Z-form) are currently known. Most structural variants of DNA can exist only under strictly controlled experimental conditions. These variants differ in 1) the number of Base Pairs per turn of the double helix; 2) the distance between base-pair planes and the angle they form with the helical axis; 3) the diameter of the helix; and 4) the handedness (right- or left-handed) of the double helix (Table 37.1).

Some of these forms interconvert upon changes in salt concentration and Hydration level. It is also possible that transitions between various structural forms of DNA occur in vivo.

Fig. 37.2. Watson-Crick model of the B-form double helix. Left: Schematic representation of the molecule (A — adenine, C — cytosine, G — guanine, T — thymine, P — phosphate, S — sugar [deoxyribose]). Right: Molecular model of DNA. (Photograph from J. D. Watson, Molecular Biology of the Gene, 3rd ed. Copyright 1976, 1970, 1965 by W. A. Benjamin, Inc., Menlo Park, Calif.)

Under physiological conditions (low salt concentration, high hydration level), the B-form is the dominant structural type of DNA. The pitch of the helix for such a molecule is 3.4 nm. A DNA turn can be visualized as two twisted stacks of "coins," with 10 coins in each stack. The stacks are held together by hydrogen bonds between opposing "coins" and wrapped by two right-handed strands of the phosphodiester backbone. Under conditions of lower hydration and higher concentrations of Na⁺ or K⁺ ions, a somewhat different structure emerges, known as the A-form. This right-handed conformation features a larger helical diameter than the B-form and a greater number of base pairs per turn. It resembles the structure characteristic of double-stranded RNA or RNA-DNA duplexes. The C–E forms are also right-handed; their formation is observed exclusively in specialized experiments and they presumably do not exist in vivo.

The Z-form of DNA is a left-handed double helix in which the phosphodiester backbone follows a zigzag trajectory along the molecular axis, hence the designation Z (zigzag) DNA.

Z-DNA is the least twisted (12 base pairs per turn) and thinnest of the naturally occurring forms, possessing only a single groove (see below). Z-DNA is detected in repeating sequences of alternating purine and pyrimidine deoxynucleotides (GC or AC) in the presence of several other stabilizing factors. These include: 1) high salt concentration or the presence of specific cations such as spermine and spermidine; 2) a high frequency of negative supercoils within the DNA molecule (see ch. 38); 3) binding of Z-DNA-specific Proteins; and 4) methylation of the carbon-5 atom in certain deoxycytidine residues.

Z-form DNA may participate in the Regulation of Gene Expression for genes located both nearby and at substantial distances from the Z-region. Certain proteins that bind to the major or minor grooves of B-form DNA are likely incapable of binding to Z-form DNA. Furthermore, the reversion of a DNA segment from the Z-form to the B-form—triggered, for instance, by the loss of methyl groups from 5-methyldeoxycytidine—can influence the torsional status of DNA regions located far from the site of reversion.

Fig. 37.3. Formation of two hydrogen bonds (dashed line) between deoxyadenosine and thymine bases (top), and three hydrogen bonds between deoxyguanosine and deoxycytidine bases (bottom). In DNA, the carbohydrate residue is 2-deoxyribose, whereas in RNA it is

D-ribose.

Torsional twisting and untwisting of DNA, much like deoxycytidine methylation, likely modulates gene activity (see below).

The presence of Z-DNA in Drosophila (fruit fly) Chromosomes has been demonstrated using Antibodies specific to the Z-form of DNA. Human DNA contains segments with the potential to transition into the Z-form, which are dispersed throughout The Genome. There is reason to believe that conditions required to stabilize the Z-form can also be realized in human cells.

Table 37.1. Characteristics of Selected DNA Structural Types

DNA Denaturation (Melting)

The double-helical structure of DNA can be "melted" in solution by increasing the temperature or lowering the salt concentration. Melting involves not only the Separation of the DNA strands but also the disruption of the stacking interactions between the nucleic acid bases within each strand. The phosphodiester bonds, however, remain unbroken. DNA denaturation is accompanied by an increase in the optical absorbance of purine and pyrimidine bases, a phenomenon known as the hyperchromic effect of DNA denaturation. Furthermore, denaturation abolishes the high viscosity characteristic of native DNA solutions, the fiber-like structure of which is maintained by both base-stacking interactions within each strand and complementary interactions between the two strands.

The separation of the strands of a given DNA molecule occurs within a specific temperature range. The midpoint of this range is referred to as the melting temperature, or $T_m$, of the DNA. The value of $T_m$ depends on the Nucleotide Composition of the DNA and the salt concentration of the solution. DNA molecules enriched in G—C pairs (which are held together by three hydrogen bonds) melt at a higher temperature than A—T-rich molecules (A—T pairs are linked by two hydrogen bonds). A tenfold increase in the concentration of monovalent cations raises the $T_m$ by 16.6°C. Formamide, commonly used in recombinant DNA experiments, destabilizes the hydrogen bonds between bases, thereby lowering $T_m$. This allows DNA or DNA—RNA hybrid strands to separate at a lower $T_m$, which reduces the likelihood of single-strand breakage that can occur at elevated temperatures.

Grooves in the DNA Structure

Upon examining the model shown in Fig. 37.2, one can observe the presence of Major and minor grooves in the DNA structure, which wind around the molecular axis parallel to the phosphodiester backbone. Within these grooves, proteins can interact specifically with particular atoms of the nucleic acid bases and thus "recognize" specific nucleotide sequences without disrupting the complementary interactions of the double helix. As will be discussed in Chapters 39 and 41, it is precisely through such interactions that regulatory proteins can control Gene Expression.

Relaxed and Supercoiled DNA

The DNA of certain organisms, such as Bacteria, Bacteriophages, and many DNA-containing animal Viruses, exists as a closed circular structure. While such a structure preserves the polarity of the molecules, it eliminates the free 3'- and 5'-hydroxyl and phosphoryl groups. Closed circles can exist in either relaxed or supercoiled forms. Supercoiling occurs when a closed circle is twisted about its own axis or when a segment of linear DNA with fixed ends is twisted. This energy-requiring process generates intramolecular structural strain. As the number of superturns increases, the internal (torsional) stress rises (this can be verified using an ordinary rubber band). Supercoils formed by left-handed twisting (opposite to the right-handed twist of the B-form DNA double helix) are termed negative. In a sense, the energy required to achieve this structural state is stored in conventional (non-negative) superturns. The energy required for a DNA molecule to transition to another type of supramolecular structure can be lowered through The formation of negatively twisted regions. One such transition is strand separation in preparation for Replication and METABOLISM/31.html">Transcription. This is why DNA Supercoiling is highly advantageous in biological systems. Enzymes that catalyze topological Changes in DNA molecules are known as topoisomerases. The most extensively studied of these is bacterial gyrase, which initiates the formation of negative supercoils.

Function of DNA

The genetic information encoded in The nucleotide sequence serves two main purposes. First, it is essential for the synthesis of protein molecules; second, it ensures the transmission of the genetic material across cellular and organismal generations. Both Functions rely on the ability of the DNA molecule to act as a template—in the first case, for transcription (recoding the information into The structure of RNA molecules), and In the second, for replication (copying the information into daughter DNA molecules).

The complementarity of the strands in the Watson-Crick double helix implies a semiconservative mode of DNA replication. This means that the strands separate, and each serves as a template for the synthesis of a new complementary sequence (Fig. 37.4). The two resulting double-helical DNA molecules, each consisting of one parental strand and one newly synthesized complementary strand, are distributed between the two daughter cells (Fig. 37.5). Thus, each daughter Cell receives genetic information identical to that of the parent cell, with each retaining one strand from the original parental DNA.

The semiconservative mechanism of replication in the bacterium Escherichia coli was definitively demonstrated in the classic experiment by Meselson and Stahl, which combined The Use of a heavy nitrogen isotope with equilibrium density gradient centrifugation.

Fig. 37.4. Double-stranded structure of DNA. Each of the two strands of the parental DNA molecule serves as a template for the synthesis of new complementary strands. (From J. D. Watson. Molecular Biology of the Gene, 3rd ed. Copyright 1976, 1970, 1965 by W. A. Benjamin, Inc., Menlo Park, Calif.)

Fig. 37.5. Expected distribution of DNA strands in semiconservative and conservative replication. Parental strands are shown in black, and newly synthesized strands in light shading. (Redrawn and reproduced with permission from Lehninger A. L. Biochemistry, 2nd ed., Worth, 1975.)

The DNA of E. coli and human DNA are chemically identical, although their nucleotide sequences differ, and human cells contain approximately 1,000 times more DNA than bacterial cells. It has been shown that the chemical mechanism of DNA replication is conserved between prokaryotes like E. coli and eukaryotes, including humans, despite differences in the specific enzymes involved in these processes in prokaryotic and Eukaryotic cells. There is every reason to believe that insights gained from studying the nucleic acid chemistry of prokaryotes are applicable to eukaryotic systems as well. Indeed, results from experiments on mammalian cells, analogous to the experiments of Meselson and Stahl, have proven consistent with the earlier data obtained for E. coli.