Fundamentals of Biochemical Engineering Part 1 - Bailey J., Ollis D. 1989

Molecular Genetics and Regulatory Systems
Molecular Genetics
DNA Replication and Mutations

Since DNA contains all the information necessary for the development and functioning of a Cell, the latter must be equipped with an extremely error-free DNA copying mechanism. During cell self-reproduction, each daughter cell must receive complete Genetic information in the form of DNA. We have already noted that the double-helical model of Introduction/20.html">DNA Structure ensures precise Replication. As a result, two identical DNA molecules are formed, each containing one strand of the original DNA. Apparently, this scheme helps minimize errors during the replication process.

In fact, the METABOLISM/36.html">DNA replication process is more complex than shown in Fig. 6.11. DNA Synthesis is a multi-step process; a special enzyme called DNA polymerase readily builds daughter strands in the 5' → 3' direction. This process occurs smoothly on one of the parental strands running in the 3'→5' direction. On the other strand, DNA polymerase synthesizes fragments of the daughter strand corresponding to the 5'→3' direction of the parental strand, while covalent joining of the fragments is carried out by another enzyme, DNA ligase. In this way, the Synthesis of the second daughter strand is also accomplished in the 3'→5' direction.

Class="center">Table 6.1. Chromosome numbers in normal Cells of various organisms

Here we will also briefly review the differences in information storage and replication between prokaryotic and eukaryotic DNA. In the poorly demarcated nucleoid of prokaryotes, there is only a single chromosome (the carrier of genetic information) containing a circular double helix of DNA. This huge molecule is 1.2 mm long, about 20 angstroms thick, and has a Molecular Weight of approximately 2.8∙10⁸. This is sufficient to encode about 2,000 different Proteins. The circular nature of E. coli DNA was first elucidated through genetic studies aimed at mapping the relative positions of individual genes. The eukaryotic chromosome is constructed from a DNA molecule associated with proteins and, possibly, a small amount of RNA.

As shown in Table 6.1, Eukaryotic cells typically contain multiple Chromosomes. Some eukaryotes, such as yeast, can be either haploid (i.e., containing only a single chromosome of each type) or diploid, with each cell containing two chromosomes of each type (except perhaps for sex chromosomes).

FIG. 6.12. Different Selection/21.html">Types of Mutations in the base-pair sequence of a DNA molecule.

A mutation is defined as A change in DNA structure that is transmitted to subsequent generations. At THE MOLECULAR LEVEL, a mutation is nothing other than an alteration in The nucleotide sequence of DNA. Some possible types of mutations are illustrated in Fig. 6.12. To some extent, mutation is a spontaneous process to which DNA is constantly subjected. However, The rate of spontaneous mutations is low, averaging about 1 error per million Gene duplications.

The severity of an error during gene replication depends on its nature. In a so-called missense mutation, the codon for one amino acid is altered in such a way that a different amino acid is inserted at the corresponding position in the protein's Amino Acid Sequence. This type of mutation leads, for example, to the abnormal Hemoglobin characteristic of individuals suffering from Sickle-Cell Anemia; in this specific case, a Glu residue is replaced by a Val residue in the abnormal protein. According to The Genetic Code (Fig. 6.3), the Glu residue is encoded by the codons GAA and GAG, whereas valine corresponds to the codons GUA and GUG. It follows that the substitution of just a single base can lead to a severe hereditary disease. Other codon changes may result, for example, in a termination codon and thus cause premature cessation of Peptide Synthesis; mutations of this type are called nonsense mutations.

Several mechanisms have been proposed for spontaneous mutations. First, the nucleotide bases of DNA can exist in several structurally distinct forms known as tautomers. It is generally assumed that the structures depicted in Fig. 2.7 predominate in native DNA; however, the existence of other tautomers cannot be ruled out, and base pairing with them could lead to errors. Another possible cause of spontaneous mutations is related to the functioning of Enzymes required for DNA SYNTHESIS AND repair (correction of structural damage). Finally, certain intermediates of normal cellular metabolism, such as peroxides, nitrous acid, and formaldehyde, are mutagens—that is, substances capable of inducing chemical mutations in DNA.

The action of Chemical Mutagens on DNA has been extensively studied by growing cells in media enriched with such agents. Mutagens include, for example, compounds whose structures closely resemble those of normal DNA bases. Because of their structural and chemical similarity, an analog rather than a normal nucleotide may be incorporated into the nucleotide chain during DNA Biosynthesis. Other types of mutagens and their mechanisms of mutagenic activity are listed in Table 6.2.

The second major cause of mutation is radiation. In particular, DNA strongly absorbs ultraviolet radiation, and at a sufficient dose, this radiation rather quickly kills most cells, while surviving cells undergo profound mutations. Every cell possesses enzymatic mechanisms for repairing DNA damaged by ultraviolet radiation. These enzymes replace the damaged segment containing covalently linked pyrimidine residues through a rather complex pathway.

In biochemical technology, The phenomenon of mutation is important from several Perspectives. Recall, for example, Fig. 1.10, which demonstrates how mutations can be used to alter The properties of a microorganism to make it more useful for a specific purpose. Mutagens and ultraviolet radiation are frequently employed to generate protist mutants with potentially higher productivity. In any effort to screen for new microbial strains, It is important to have efficient Methods for identifying and isolating mutants with specific properties. Table 6.3 lists some of the primary methods used for this purpose. Other approaches to the Genetic Transformation of producer organisms will be discussed in Sections 6.2 and 6.3.

Table 6.2. Chemical mutagens and their corresponding mechanisms of mutagenic activity^a

^a From Stanier, R. Y., Doudoroff, M., Adelberg, E. A., The Microbial World, p. 418, Prentice-Hall, Inc., Englewood Cliffs, N. J., 1970.

On the other hand, mutations can also pose certain difficulties in biotechnology. The success of an industrial microbiological process often requires pure microbial strains with well-characterized properties. At the same time, the possibility of mutations in such cultures can never be entirely ruled out, making regular monitoring of their genetic homogeneity essential. Other practical Problems associated with mutations and other types of genetic instability will be discussed in subsequent chapters.