BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

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

24.23. The Complexity of the Replication Apparatus Appears Necessary to Ensure Extremely High Fidelity

Current models of the Molecular Mechanism of METABOLISM/36.html">DNA Replication in E. coli are illustrated in Fig. 24.44 and Table 24.2. What is striking is the sheer complexity of the interactions among the numerous Proteins involved in this process. Genetic analysis reveals that at least 15 proteins participate directly in DNA replication. Why is the DNA replication machinery so complex? In particular, why does DNA Synthesis begin with An RNA primer that is subsequently removed? If DNA polymerases could initiate chain synthesis de novo, an RNA primer would be unnecessary. However, such a capability would be incompatible with the extraordinarily high fidelity of DNA polymerases. Recall that before forming a new phosphodiester bond, DNA polymerases check the correctness of the preceding base pair. This proofreading function significantly reduces the error rate. RNA polymerases, by contrast, can initiate chain synthesis de novo because they do not check

the preceding base pair. Their error rate is several orders of magnitude higher than that of DNA polymerases. An ingenious solution to this problem was found: initiate DNA synthesis with a polynucleotide synthesized with low fidelity, but mark its "temporary" nature by incorporating ribonucleotides into its composition. DNA polymerase I then excises these short RNA primer sequences and replaces them with a DNA sequence synthesized with high fidelity. It appears that many of the details that complicate the DNA Replication Mechanism are designed to ensure extraordinary precision. Genetic analysis indicates an error rate of one per 10⁹-10¹⁰ Base Pairs read.

Class="center">Fig. 24.44. Schematic diagram of enzymatic processes at the E. coli Replication fork. Fragments highlighted in blue catalyze the initiation, elongation, and joining (via DNA ligase) of DNA strands

Table 24.2. E. coli Replication Proteins

24.24. DNA Damage is Constantly Repaired

Because numerous chemical and physical agents cause damage to DNA, all Cells possess specialized mechanisms to repair such lesions. Bases in DNA can be modified or lost, phosphodiester bonds in the backbone can be broken, and the two strands can become cross-linked. These lesions are induced by ionizing radiation, ultraviolet light, and various chemical agents. Many types of DNA damage can be successfully repaired because the Genetic information is stored in both strands of The Double Helix. Consequently, information lost from one strand can be retrieved from the intact complementary strand.

One of the best-understood repair mechanisms is pyrimidine dimer excision (Fig. 24.45), which occurs when DNA is exposed to ultraviolet light. Adjacent pyrimidine residues in a single DNA strand can form covalent cross-links under these conditions. Such a pyrimidine dimer distorts the double helix, blocking both replication and Gene Expression until the lesion is removed.

Fig. 24.45. Model of a uracil dimer formed by ultraviolet irradiation. A thymine dimer has a structurally similar configuration

Four enzymatic activities play a crucial role in executing this process (Fig. 24.46). The first of these, a UV-specific endonuclease, locates the damage and introduces a single-strand break near the dimer, typically on the 5'-side. The region containing the dimer protrudes from the double helix, allowing DNA polymerase I (or another similar polymerase) to carry out repair synthesis in the 5' → 3' direction. The 3'-end of the broken strand serves as the primer, while the intact complementary strand acts as the template. Next, the region housing the pyrimidine dimer is excised by the 5' → 3' nuclease activity of the DNA polymerase. Finally, the newly synthesized strand and the remaining DNA strand are joined by DNA ligase. Another repair pathway is the photochemical Cleavage of the pyrimidine dimer. Nearly all cells contain a photoreactivating enzyme that recognizes the dimer and splits it back into the original bases using the energy of absorbed blue light.

Fig. 24.46. Repair of a DNA region containing a thymine dimer via the sequential action of a specific endonuclease, DNA polymerase, and DNA ligase. The thymine dimer is shown in blue, and the newly synthesized DNA patch in red

24.25. Skin Cancer in Xeroderma Pigmentosum Results from Defective DNA Repair

Xeroderma pigmentosum is a rare human Skin disorder inherited as an autosomal recessive trait. In homozygous affected individuals, the skin is extremely sensitive to sunlight and ultraviolet radiation. Severe skin damage manifests already in childhood and becomes progressively more severe with age. The skin becomes dry, and the dermis undergoes significant atrophy. Keratoses develop, the eyelids become scarred, and the cornea is affected. Skin Cancer frequently arises at multiple sites. Many patients die before reaching the age of 30 due to metastasis of these malignant skin tumors.

Xeroderma — from Greek words meaning "dry skin." This term was first introduced by F. Hebra and M. Kaposi in 1874 to describe the "parchment skin" and abnormal pigmentation observed in one of their patients.

In human DNA, just as in E. coli, ultraviolet light induces The formation of pyrimidine dimers. Moreover, the repair mechanisms in humans and E. coli appear to be similar. Studies of skin fibroblasts from patients with xeroderma pigmentosum have revealed that one form of this disease is accompanied by a biochemical defect. In normal fibroblasts, half of the pyrimidine dimers generated by UV irradiation are excised in less than a day. In contrast, fibroblasts obtained from xeroderma pigmentosum patients show almost no dimer excision over the same time period. Which step in the repair process is impaired? The answer was obtained by determining the Molecular Weight of DNA strands from UV-irradiated fibroblasts. In normal cells, a marked decrease in the molecular weight of single-stranded DNA occurs within a few hours after irradiation. This reduction in molecular weight results from the initial reaction of the repair process—specifically, the cleavage of the DNA strand adjacent to the pyrimidine dimer. Following UV irradiation of xeroderma pigmentosum cells, however, no such decrease in molecular weight is observed. Consequently, this skin disorder is likely caused by the inactivation of the endonuclease that hydrolyzes the DNA backbone next to the pyrimidine dimer. The severe clinical consequences of this enzymatic defect underscore the critical importance of DNA Repair processes.