Biochemistry: The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
Genetic Methods
Types of Mutations

It is difficult to overestimate Structure/19.html">The Importance of Genetic Methods in shaping our modern understanding of molecular biology. Therefore, it is essential for biochemists to be familiar with these techniques. Biochemical literature is increasingly saturated with genetic terminology. More importantly, however, genetic methods are now being used to investigate A wide variety of complex biochemical phenomena. Furthermore, looking ahead, we clearly perceive the need to understand issues related to Mutations and Gene Variability.

Alterations in Introduction/20.html">DNA Structure occur very infrequently. For instance, on average, a gene may undergo 10⁶ replications before a noticeable mutation takes place [128a]. Nevertheless, by working with Bacteria or Bacteriophages, we can examine an extremely large number of individuals in search of mutations. If, for example, one million Viral Particles are plated on an Agar plate under conditions that allow the identification of a specific gene mutation, we can reasonably expect to find one mutant on average. The most common mutations are those caused by base-pair substitutions (point mutations). These occur As a result of the incorporation of an incorrect base during METABOLISM/36.html">DNA Replication or repair. In such mutations, a single base in a codon triplet is replaced by another. This results in a different codon, leading to the substitution of one amino acid for another in the corresponding protein¹⁾. The replacement of one pyrimidine by another (C—>T or T—>C) or one purine by another is sometimes referred to as a transition, whereas the replacement of a purine by a pyrimidine or, conversely, a pyrimidine by a purine is called a transversion.

¹⁾ The alteration of a single base does not necessarily lead to an amino acid substitution due to the degeneracy of The Genetic Code, i.e., the fact that a single amino acid can be encoded by multiple codons.

Transitions occur significantly more frequently than transversions. One possible cause of such mutations is the pairing of one of the bases with a minor tautomer (Ch. 2, Sec. D, 7)^!). For instance, A can pair with a minor tautomer of C, inducing a T-to-C substitution. It should be noted that the appearance of an incorrect base in one strand can lead to the subsequent replication—via correct pairing in one of the double strands of the daughter DNA—of a GC pair instead of an AT pair, or vice versa.

Based on the observed frequency of point mutations (one mutation per 10⁶ gene replications), we can calculate that there is one mutation per 10⁹ single-nucleotide replications. Point mutations tend to be reversible, with back-mutations often occurring at rates comparable to those of forward mutations. This implies that in one out of every 10⁹ "reverse" events, the exact same nucleotide will mutate, thereby restoring the gene to its original state. This phenomenon is readily explained. For example, if T is replaced by C—since C forms a minor tautomer and pairs with A—the mutation will result in the appearance of a GC pair in the DNA double helix of the progeny. During the replication of this pair, There is a small yet definite probability that the C in the parental DNA strand will once again adopt a minor tautomeric structure and pair with A rather than G, which in turn leads to a back-mutation.

Although The rate of spontaneous mutations is low, it can be significantly increased by the action of Chemical Mutagens (Sec. 3.1) or radiation. This approach has made it possible to easily measure the rates of both forward and reverse mutations. Once such measurements were carried out, it became apparent that while mutations induced by certain chemical compounds, such as acridine Dyes, can be reverted, the frequency of such reversion is markedly lower than that of conventional back-mutations. It has been shown that these mutations result either from the deletion (loss) of one or more NUCLEOTIDES from the chain or from the insertion (addition) of extra nucleotides. Deletion and insertion mutations apparently arise from errors During genetic recombination and the Repair of Damaged DNA strands.

Mutations resulting from the deletion or insertion of one or more nucleotides are termed frameshift mutations. Consider an RNA transcribed from a DNA molecule that has undergone a deletion or insertion. The Messenger RNA is read by the protein-synthesizing machinery starting from a fixed initiation point. As codons—each consisting of three bases—are read, Amino Acids are incorporated into the protein in the order dictated by the corresponding codons. However, if a deletion or insertion is encountered in the DNA (and consequently in the mRNA), all subsequent codons will be read incorrectly because the reading frame will be shifted forward or backward by one or two nucleotides^{1, 2)}. As a result, the synthesized protein will bear little resemblance to the one produced in the non-mutant Organism; typically, such Proteins turn out to be completely devoid of functional activity. Nevertheless, a frameshift near the 3'-end of a gene can cause a readthrough of the termination codon, leading to the synthesis of a functionally active protein with an extended C-terminus. It is believed that several types of Hemoglobins may have arisen through this very mechanism [129a, b].

¹⁾ There is a possibility that a purine base may pair with another purine base if the latter is in the energetically less favorable syn-conformation (Ch. 2, Sec. D, 4) [129a].

²⁾ In cases where the reading frame is shifted by three nucleotides, a normal protein is produced, which, however, either lacks a single amino acid residue or contains an extra residue.

Frameshift mutations are classified as either + or − depending on whether an insertion or a deletion of a small number of bases has occurred. Thus, insertion mutations can be designated as +1, +2, etc. Mutations also occur in which large segments of DNA are gained or lost. For instance, a major insertion may take place via the incorporation of a long stretch of foreign DNA into a gene. The loss or addition of large chromosomal fragments can likewise be categorized as deletion or insertion mutations.