Introduction to Molecular Biology: From Cells to Atoms - Anthony Rees, Michael Sternberg 2002

Nucleic Acids and Genes
DNA Structure

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Fig. 18.1. The DNA double helix.

DNA—deoxyribonucleic acid—is a biological macromolecule that serves as the carrier of Genetic information in all eukaryotic and Prokaryotic Cells, as well as in many Viruses.

Research on pneumococci conducted by Avery, MacLeod, and McCarty in 1944 provided compelling evidence that DNA is indeed the genetic material. They studied two bacterial forms: an infectious (virulent, designated as S) strain that forms smooth colonies on Agar, and a mutant, non-virulent (designated as R) strain that forms rough colonies. Avery and his colleagues demonstrated that DNA extracted from heat-killed S Bacteria is capable of transforming the non-virulent R form into the virulent form, and that the virulence of these transformed R bacteria is heritable across subsequent generations. This clearly indicated that DNA carries genetic information.

Studies on the T2 bacteriophage conducted by Hershey and Chase in 1952 yielded further evidence regarding the genetic role of DNA. These authors sought to determine which component of the virus—DNA or protein—is responsible for infecting E. coli bacteria. They grew the virus in the presence of radioactive isotopes ³²P (to label the DNA) and ³⁵S (to label the protein) and used it to infect E. coli. The results showed that the infecting agent molecules contained ³²P. Thus, it was demonstrated that the information required for The formation of new Viral Particles resides within the viral DNA.

The Double Helix: A MODEL OF DNA Structure was proposed by Watson and Crick in 1953. According to this three-dimensional model, the DNA molecule consists of two polynucleotide chains wound around a common axis in a right-handed helix¹—hence the term double helix. The two chains run in opposite directions. The sugar-phosphate backbone forms the outer periphery of the double helix, while the nitrogenous bases are stacked on the inside with their planes perpendicular to the helical axis.

¹ More precisely, a screw axis. — Ed. note

Specific Hydrogen Bonds form between the bases, resulting in what is known as Watson-Crick base pairing. Adenine always forms hydrogen bonds with thymine, and guanine with cytosine. Consequently, the bulkier Purines always pair with the smaller Pyrimidines. This ensures that the distance between the C1' atoms of deoxyribose in the two chains remains identical for both AT and CG pairs, measuring 1.085 nm. As a result, AT and GC pairs fit into the double helix without causing any significant distortion of backbone geometry.

Complementarity refers to the mutual correspondence of base sequences in opposite DNA strands. If one strand has adenine at a given position, the complementary strand must have thymine at that same position, and vice versa, allowing specific hydrogen bonds to form between the bases. Similarly, if one strand contains guanine, the opposing strand must contain cytosine, and vice versa. Complementarity is vital for METABOLISM/36.html">DNA Replication (Chapters 20 and 21).

The base composition ratios in DNA, established by Chargaff and his coworkers in the 1950s, played a crucial role in the formulation of the double helix model. Chargaff discovered that in DNA from A wide variety of sources, The amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine (i.e., A ≡ T and G ≡ C). These equalities are a direct consequence of selective base pairing.

The geometry of the double helix is such that adjacent Base Pairs are spaced 0.34 nm apart and rotated by 36° relative to one another around the helical axis. Consequently, each turn of the helix comprises 10 base pairs (360° / 36° = 10), and the pitch of the helix is 3.4 nm (10 × 0.34 nm). The diameter of the double helix is approximately 20 nm. The DNA double helix features grooves, which arise because the sugar-phosphate backbone is situated further from the helical axis than the bases. There are two such grooves: a major groove and a minor groove.

X-Ray Diffraction patterns of DNA fibers obtained by Franklin and Wilkins between 1950 and 1953 provided critical data for constructing the double helix model. The idealized diffraction pattern (Fig. 18.2) displays a characteristic cross-like arrangement of reflections (spots) resulting from the regular structure of DNA. The distance between layer lines corresponds to a period of 3.4 nm—the pitch of the double helix—while a strong reflection on the 10th layer line corresponds to a period of 0.34 nm, representing the distance between adjacent base pairs. These parameters characterize the B-form of DNA (see below).

Fig. 18.2. Idealized X-ray diffraction pattern of a DNA fiber. The analysis of these measurements led to The Development of the DNA model.

The Stability of the double helix is maintained by a combination of interactions. Hydrogen bonds between the bases contribute to this stability, but base stacking (interplanar interactions) appears to play an even more crucial role. Stacking not only ensures favorable Van der Waals contacts between atoms but also provides additional stabilization through the overlap of π-orbitals from adjacent bases. Further stabilization is afforded by the hydrophobic effect, which shields the non-polar bases from direct contact with the aqueous solvent. Conversely, the sugar-phosphate backbone, with its polar groups and charged atoms, is exposed to the solvent, which also helps stabilize the structure.

DNA polymorphism refers to the ability of the double helix to adopt various Conformations. X-ray crystallographic studies of polynucleotide crystals¹ have revealed three primary structural types: the A-, B-, and Z-forms. B-DNA is the standard Watson-Crick structure, in which the planes of the base pairs are perpendicular to the helical axis. In A-DNA, the base pair planes are tilted by approximately 20° relative to the normal of the right-handed helical axis, with 11 base pairs per turn. A-DNA is formed upon dehydration of B-DNA fibers. Z-DNA is a left-handed helix containing 12 base pairs per turn; the letter 'Z' reflects the zig-zag conformation of the sugar-phosphate backbone. The base planes remain approximately perpendicular to the axis. In vivo, DNA predominantly exists in the B-form, though specific segments may adopt A-, Z-, or other alternative conformations¹.

¹ Short polynucleotides, known as oligonucleotides. — Ed. note

Determining The nucleotide sequence of DNA (sequencing) is now a rapid process, and this major breakthrough has enabled molecular biologists to tackle the crucial challenge of determining Gene structure (Chapters 26–28). Two main sequencing Methods are widely used: the Maxam-Gilbert chemical Cleavage method and the Sanger chain-termination method. Each of these methods is typically limited to sequencing DNA fragments only a few hundred bases long. Therefore, the initial step involves cleaving long chains (such as entire Chromosomes) into manageable fragments. This is accomplished using restriction Enzymes (Restriction Endonucleases). Restriction enzymes are isolated from bacteria and fall into two main types. Only Type II enzymes are used for sequencing; they cleave (cut) double-stranded DNA at specific base sequences, usually 4 to 6 base pairs in length (Chapter 30). There are numerous restriction enzymes, each specific to a particular base sequence. For example, the enzyme EcoRI from E. coli cleaves sequences at the sites indicated by the arrows:

The key feature of base sequences cleaved by restriction enzymes is that they are typically palindromes: the sequences of both strands read identically from the 5' to the 3' end. Such sequences possess a 2-fold axis of Symmetry, indicated in the figure by the ♦ symbol.

¹ The Formation of the Z-form, cruciform structures, and other conformations is promoted by DNA Supercoiling. — Ed. note

In the chemical cleavage method, the 5' end of the DNA molecule is first labeled with radioactive ³²PO₄ using the enzyme polynucleotide kinase. The DNA is then treated with specific Reagents that cleave the chain near particular bases. This cleavage is carried out in four parallel reactions under conditions where a break occurs after A in one reaction, after G In the second, after C in the third, and after both C and T in the fourth. The reaction conditions are controlled to yield a mixture of fragments of all possible lengths, extending from the labeled 5' end to each position where a given base occurs. For instance, if G is located at positions 1, 5, 7, and 19 in a 20-base fragment, one of the degradation mixtures will contain radioactive fragments of 5, 7, and 19 bases in length, along with free G. Using Polyacrylamide gel Electrophoresis, each of the four mixtures is separated into fragments of varying lengths. The gel is then exposed to photographic film, producing dark bands where the radioactive material is located (a process known as autoradiography). The base sequence can be read directly from the film, as illustrated, for example, in Fig. 19.3.

The chain-termination method employs a different approach. It is based on the synthesis of a radioactively labeled complementary DNA strand using native DNA AS A template (Chapters 20 and 21). Synthesis is catalyzed by DNA polymerase I (Chapters 20 and 21). The reaction mixture contains four unlabeled deoxynucleoside triphosphates required to build the complementary chain, along with a modified form of one of the bases (its 2',3'-dideoxy analog), the incorporation of which halts further chain elongation. As a result, DNA chains of varying lengths are synthesized. Four separate reaction mixtures are used, each containing the four standard monomers plus one specific chain-terminating base analog. The resulting chains from each mixture are then separated by gel electrophoresis and subjected to autoradiography. The base sequence is read directly from the resulting autoradiogram. Sanger's method was developed as an advancement over the earlier 'plus-minus' method, which also relied on the synthesis of complementary DNA strands.