BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

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

24.26. DNA Contains Thymine Instead of Uracil, Which Makes the Repair of Deaminated Cytosine Possible

Cytosine in DNA spontaneously deaminates at a measurable rate to form uracil.

The deamination of cytosine is potentially mutagenic because the resulting uracil pairs with adenine, and consequently, one of the daughter strands will contain an AU base pair instead of the original GC pair:

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This mutation is corrected by a repair system that recognizes foreign uracil in the DNA molecule (Fig. 24.47). First, uracil-DNA glycosylase hydrolyzes the glycosidic bond between the uracil residue and deoxyribose. At this stage, the DNA backbone remains intact, but a single base is missing. Then, a specific endonuclease recognizes this defect and cleaves the backbone adjacent to the missing base. DNA polymerase I excises the remaining deoxyribose phosphate and inserts cytosine, which is complementary to guanine in the undamaged strand. Finally, the repaired strand is sealed by DNA ligase.

Fig. 24.47. Uracil residues in DNA are excised and replaced by cytosine, the original base

For many years, it remained a mystery why DNA contains thymine rather than uracil, given that both bases pair with adenine. The only difference between them is the methyl group of thymine in place of the hydrogen atom at C-5 in uracil. Why is this methylated base used in DNA but not in RNA? Recall that the methylation of deoxyuridylate to form deoxythymidylate requires a large expenditure of energy (Sec. 22.16). The relatively recent discovery of an active system that repairs the deamination of cytosine provides a compelling answer to this puzzle. Uracil-DNA glycosylase does not remove thymine from DNA. Thus, the methyl group of thymine serves as a tag that distinguishes it from deaminated cytosine. Without this tag, a correctly positioned uracil would be indistinguishable from a uracil generated by deamination. The defect would go unnoticed, and a mutagenic substitution of a GC pair for an AU pair would inevitably occur in one of the daughter DNA molecules. The repair system that searches out uracils and leaves thymines suppresses such Mutations. In all likelihood, thymine is used in DNA instead of uracil to enhance The fidelity of Genetic information. In contrast, RNA is not repaired and utilizes uracil because it represents a less costly building block.

24.27. Restriction Endonucleases Revolutionized DNA Analysis

Restriction Enzymes are endonucleases capable of recognizing specific base sequences in double-stranded DNA and cleaving both strands. These exceptionally precise scalpels are a magnificent gift of nature to biochemists. The existence of restriction enzymes has made it possible to conduct experiments that were unimaginable only a few years ago. They are indispensable tools for investigating Chromosome Structure, determining nucleotide sequences in very long DNA molecules, isolating genes, and generating new DNA molecules for cloning. The Development of all these techniques was initiated by Werner Arber, Hamilton Smith, and Daniel Nathans.

A palindrome — a word, sentence, or verse that reads the same forwards and backwards.

Examples:

Radar

A man, a plan, a canal: Panama.

Was it a car or a cat I saw?

Madam, I'm Adam.

Roma tibi subito motibus ibit amor.

Derived from the Greek word *palíndromos*, meaning running back.

Restriction Endonucleases are found in A wide variety of prokaryotes. The Biological Role of these enzymes is to cleave foreign DNA molecules. The host's own cellular DNA is not cleaved because the sites recognized by its restriction enzymes are methylated. The relationship between Restriction and modification is discussed in Chap. 30 (Sec. 30.9). Crucially, many restriction enzymes recognize specific DNA sequences four to six Base Pairs long and hydrolyze the phosphodiester bonds in both strands within this region. A striking feature of such Cleavage sites is their twofold rotational Symmetry. In other words, the recognized base-pair sequence is a palindrome.

The cleavage sites are located symmetrically about the twofold axis. For example, the restriction enzyme from Streptomyces achromogenes recognizes the sequence

In each strand, this endonuclease cleaves the phosphodiester bond between G and C that is distal to the axis of symmetry. To date, more than 80 restriction enzymes have been purified and characterized. Their names consist of a three-letter abbreviation for the microorganism (e.g., Eco from E. coli, Hin from Haemophilus influenzae, Hae from H. aegyptius), followed by the strain designation and a Roman numeral. The Specificity of certain enzymes is shown in Fig. 24.48. Note that the breaks produced in the two strands may be either staggered relative to each other or directly opposite one another.

Fig. 24.48. Specificity of selected restriction endonucleases. The base-pair sequences recognized by these enzymes possess a twofold axis of symmetry. The two DNA strands in this region are symmetrical about the axis indicated by the green oval (one strand is rotated by 180° relative to the other). Cleavage sites are indicated by red arrows. The abbreviated name of each restriction enzyme is given to the right of the sequence it recognizes.

Restriction enzymes are used to cleave DNA molecules into specific fragments that are much easier to analyze and manipulate than the original molecule. For example, EcoRI cleaves the circular double-stranded DNA of the SV-40 virus (5.1 kb in length) at only one site, HpaI at four sites, and HindII at 11 sites. A DNA segment generated by one restriction enzyme can be specifically cleaved into smaller fragments using another enzyme. By employing multiple restriction enzymes, Chromosomes can be mapped (Section 31.8). Furthermore, the set of fragments produced by restriction enzymes can serve as a distinctive "fingerprint" for a given DNA molecule. Minor differences between similar DNA molecules are readily detected by electrophoretic Separation of their restriction fragments. For any given gel, the electrophoretic mobility of a DNA fragment is inversely proportional to the logarithm of the number of base pairs (up to a certain fragment length limit). Polyacrylamide gels are used to separate DNA fragments up to 1000 base pairs long, whereas the more porous agarose gels are used for longer molecules. If the DNA is radioactively labeled, the bands can be detected by autoradiography. Alternatively, the gel can be stained with ethidium bromide, which fluoresces brightly orange upon binding to double-helical DNA. Using this method, a band containing as little as 50 ng of DNA is easily visualized (Fig. 24.49). In addition to sensitivity, a major advantage of such gels is their high resolving power.

Fig. 24.49. Electrophoretic separation of fragments generated by Digestion of SV-40 DNA with three different restriction enzymes. These fragments fluoresce due to staining of the gel with ethidium bromide.