BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

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

24.7. Certain Viruses Contain Single-Stranded DNA at Specific Stages of Their Life Cycle

DNA is not always a double-stranded molecule. Robert Sinsheimer discovered that the DNA of phage ɸX174, a small virus that infects E. coli, is a single-stranded molecule. Several experimental facts led him to this unexpected Conclusion. First, the base composition of phage ɸX174 DNA does not obey Chargaff's rules where [A] = [T] and [G] = [C]. Second, the viscosity of a ɸX174 DNA solution is significantly lower than that of an E. coli DNA solution at the same concentration. The hydrodynamic properties of ɸX174 DNA correspond to a polymer in a random-coil conformation. In contrast, double-helical DNA behaves hydrodynamically like a very rigid rod. Third, the amino groups of the bases in ɸX174 DNA readily react with formaldehyde, whereas the bases in double-helical DNA are almost inaccessible to this reagent.

The discovery of this single-stranded DNA cast doubt on the universality of the semiconservative Replication model proposed by Watson and Crick. However, it was soon shown that ɸX174 DNA exists in a single-stranded state only during a portion of the viral life cycle. Sinsheimer found that infected E. coli Cells contain a double-stranded form of ɸX174 DNA. This double-stranded DNA is called the replicative form because it serves as a template for the synthesis of progeny viral DNA. Viruses containing single-stranded RNA also replicate via an intermediate double-stranded replicative form. The mechanisms of these processes are discussed in more detail in Chapter 30. Here, It is important only to emphasize that these studies confirmed the generality of the Watson-Crick replication scheme. Double-stranded DNA (or RNA) is the replicative form for all known genes.

Class="center">Fig. 24.17. Electron micrograph of ɸX174 virus particles

24.8. DNA Molecules Are Very Long

Before examining The Mechanism of METABOLISM/36.html">DNA replication, which is a complex enzymatic process, let us consider some of The properties of DNA. A striking feature of naturally occurring DNA molecules is their extraordinary length. The E. coli chromosome is a single molecule of double-helical DNA containing 4 million Base Pairs. The mass of this DNA molecule is 2.6 • 10⁶ kDa. It has an extremely asymmetric shape: its contour length is 14 • 10⁶ Å and its diameter is 20 Å. The contour length (1.4 mm) of this DNA molecule corresponds to the dimensions of macroscopic structures, whereas its diameter of 20 Å is on the scale of atomic dimensions. Bruno Zimm showed that the largest chromosome in Drosophila melanogaster contains a single DNA molecule consisting of 6.2 • 10⁷ base pairs. The contour length of this molecule is 2.1 cm. Such highly asymmetric DNA molecules are extremely susceptible to breakage by shear forces. Unless special precautions are taken during handling, they are easily broken into fragments whose mass is 1000 times smaller than that of the original molecule.

Images of DNA molecules from many Bacteria and viruses have been directly obtained using an Electron microscope (Fig. 24.18). The dimensions of some such DNA molecules are given in Table 24.1.

Fig. 24.18. Electron micrograph of a bacteriophage DNA molecule (RFII form)

Table 24.1. Dimensions of Some DNA molecules

It should be noted that even the smallest DNA molecules are highly elongated. For example, polyoma virus DNA contains 5100 base pairs and has a contour length of 1.7 µm (17000 Å). Recall that the diameter of a Hemoglobin molecule is 65 Å, and the length of Collagen, one of the longest Proteins, is 3000 Å. This comparison highlights the immense length and pronounced Asymmetry of DNA molecules.

To measure the lengths of nucleic acid molecules, a unit of length equal to 1000 nucleotide pairs for double-stranded nucleic acid molecules (kb, or kilobase pairs) or 1000 NUCLEOTIDES for single-stranded molecules (nt, or kb) is used. 1 kb of double-stranded DNA has a contour length of 0.34 µm and a mass of approximately 660 kDa.

24.9. The Double Helix Can Be Reversibly Melted

The two strands of the DNA helix can readily separate if the Hydrogen Bonds holding the paired bases together are disrupted. This can be achieved either by heating the DNA solution or by adding acid or alkali to the solution to ionize the bases. The unwinding of The Double Helix is called melting because it occurs abruptly within a narrow Temperature range. The melting temperature (T_m) is the temperature at which half of the helical Structure is disrupted. The sharpness of this transition indicates that the DNA double helix is a highly cooperative structure. DNA melting is easily monitored by measuring its absorbance at 260 nm. The disruption of base-stacking interactions leads to an increase in absorbance—an effect known as hyperchromism (Fig. 24.19).

Fig. 24.19. Upon melting of the double helix as it transitions to a single-stranded form, the absorbance of the DNA solution at 260 nm increases

The melting temperature of a DNA molecule depends significantly on its nucleotide composition. DNA molecules rich in GC base pairs have higher T_m values than those rich in AT base pairs (Fig. 24.20). In general, T_m

Fig. 24.20. Melting curves of various DNAs. The graph plots relative absorbance at 260 nm versus temperature (absorbance at 25°C is set to 1). T_m is 69°C for E. coli DNA (50% GC pairs) and 76°C for P. aeruginosa DNA (68% GC pairs)

The melting temperature ($T_m$) of DNA from many species increases linearly from 77 to 100°С as the GC content increases from 20 to 78%. GC pairs are more stable than AT pairs because their bases are held together by three hydrogen bonds rather than two. Consequently, AT-rich regions of DNA melt first (Fig. 24.21). As we will see below, the double helix is unwound in vivo through the action of specialized proteins, some of which drive unwinding by hydrolyzing ATP (Section 24.21).

Fig. 24.21. Electron micrograph of a DNA molecule partially unwound by alkali Treatment. The single-stranded regions appear as loops that stain less intensely than the double-stranded segments. These unwound DNA regions are rich in AT base pairs. One of them is indicated by an arrow

The separated complementary DNA strands spontaneously reassociate to reform a double helix when the temperature drops below $T_m$. This renaturation process is sometimes referred to as annealing. The rate of reassociation depends on the concentration of complementary sequences (Section 29.10). The ease with which Double helices melt and reassociate is critical for DNA to perform its biological Functions.