BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 26. THE GENETIC CODE AND THE RELATIONSHIP BETWEEN GENES AND PROTEINS

26.13. Mutations Result from Alterations in DNA Base Sequences

There are several Selection/21.html">Types of Mutations: 1) substitution of one base pair (or several pairs) by another; 2) deletion of one or more Base Pairs; 3) insertion of one or more base pairs (Fig. 26.11). The most common type of mutation is the replacement of one base pair by another. The spontaneous mutation rate in bacteriophage T4 has been estimated at 1.7 • 10^-8 per base pair per Replication cycle. For E. coli and Drosophila melanogaster, these values are 4 • 10^-10 and 7 • 10^-1, respectively.

Class="center">Fig. 26.11. Various types of mutations can be illustrated using typos as an analogy

There are two types of single base-pair substitutions. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. Conversely, a transversion is the substitution of a purine by a pyrimidine or a pyrimidine by a purine:

A mechanism for the spontaneous occurrence of transitions was proposed by Watson and Crick in their classic paper on the DNA double helix. They noted that certain hydrogen atoms of each of the four bases can shift their positions. Such tautomeric forms, which arise with low probability, can form

unusual base pairs distinct from A-T and G-C. For example, the rare imino tautomer of adenine can pair with cytosine (Fig. 26.12). In the next replication cycle, this adenine may revert to its normal tautomeric state and pair with thymine, whereas the cytosine will pair with guanine. As a result, one of the daughter DNA molecules will contain a G-C pair instead of an A-T pair (Fig. 26.13).

Fig. 26.12. A rare tautomeric form of adenine pairs with cytosine instead of thymine. This tautomer is formed by the shift of a proton from the 6-amino group to the N-1 atom

Fig. 26.13. Pairing of the rare tautomeric form of adenine (A*) with cytosine (C) leads to the appearance of a G—C pair in the next generation

In addition, mutations can be caused by defective DNA polymerases. An E. coli mutant exists in which the mutation rate is 1000 times higher than normal, likely due to an altered polymerase. A distinctive feature of mutations in these Cells is that almost all of them are A-T → G-C transversions.

26.14. Some Chemical Mutagens Are Highly Specific

Base analogs, such as 5-bromouracil and 2-aminopurine, can be incorporated into DNA. They induce transitions by disrupting base-pairing rules during their own incorporation into DNA or in the subsequent replication cycle (Fig. 26.14). The thymine analog 5-bromouracil normally pairs with adenine. However, the proportion of the enol tautomer in 5-bromouracil is higher than in thymine, possibly due to the high electronegativity of the bromine atom compared to the methyl group at C-5. The enol form of 5-bromouracil pairs with guanine, which induces A-T → G-C transitions. 2-Aminopurine normally pairs with thymine. Unlike adenine, the common tautomer of 2-aminopurine can form a single Hydrogen bond with cytosine. Therefore, 2-aminopurine is capable of inducing A-T → G-C transitions.

Fig. 26.14. The thymine analog 5-bromouracil occasionally pairs with guanine instead of adenine. The presence of a bromine atom at C-5 increases the proportion of the rare tautomer formed by the shift of a proton from N-3 to the oxygen atom at C-4

The action of other mutagens is based on chemical modifications of DNA bases. For example, nitrous acid reacts with bases containing an amino group, causing Oxidative Deamination of adenine to hypoxanthine, cytosine to uracil, and guanine to xanthine. The resulting hypoxanthine pairs with cytosine rather than thymine, and uracil pairs with adenine rather than guanine. Xanthine, like guanine, pairs with cytosine. Consequently, nitrous acid induces A-T → G-C transitions. Hydroxylamine (NH₂OH) is a highly specific mutagen. It reacts almost exclusively with cytosine to yield a derivative that pairs with adenine rather than guanine. Hydroxylamine induces a transition in only one direction: G-C → A-T.

Another type of mutation is caused by flat aromatic molecules, such as acridines. These compounds intercalate into DNA, meaning they slip between adjacent base pairs in the DNA double helix (Section 25.18). Intercalators apparently stabilize slipped-strand base pairing in repetitive sequences, such as CGCGCGCG. As a result, they cause insertions or deletions of one or more base pairs. The Effect of such mutations is a frameshift, unless the total number of deleted or inserted bases is a multiple of three. It was precisely the analysis of such mutants that proved the triplet nature of METABOLISM/28.html">The Genetic Code.

26.15. Many Mutagenic Carcinogens Can Be Detected by Their Mutagenic Activity in Bacteria

Many human tumors arise as a result of exposure to toxic chemicals. Since these chemical carcinogens are typically mutagenic, it is believed that DNA damage underlies both carcinogenesis and mutagenesis. It is important to identify these compounds and assess their potential biological activity in order to minimize their impact on humans. Bruce Ames developed a simple and sensitive test for detecting Chemical Mutagens. A thin layer of Agar containing about 10⁹ cells of a specially constructed Salmonella test strain is poured onto a Petri dish. These Bacteria are unable to grow in the absence of Histidine because they carry a mutation in one of the genes for The Biosynthesis of this amino acid. Adding a mutagen to the center of the plate induces the appearance of numerous new mutations. A small fraction of these mutations results in a reversion of the original mutation (a return to the wild type), so that the cells acquire The ability to synthesize histidine. These revertants multiply in the absence of exogenous histidine and appear as distinct colonies on the plate after incubation at 37°C for two days (Fig. 26.15). For example, 0.5 µg of 2-aminoanthracene yields 11,000 revertant colonies compared to only 30 spontaneous revertants in the absence of the mutagen. Experiments with various concentrations of the test compound can be easily set up to obtain a dose-response curve. This relationship is typically linear, which implies that there is no threshold concentration in mutagenesis.

Fig. 26.15. Salmonella mutagenicity test. A. Approximately 10⁹ histidine-dependent bacteria are plated onto an agar plate. B. A filter paper disk impregnated with a mutagen is placed on the plate. As a result, numerous histidine-synthesizing revertants appear, forming a halo of colonies around the white disk. The small number of colonies on plate A represents spontaneous revertants.

Some test strains are sensitive to base-pair substitutions, whereas others can be used to detect base-pair deletions or insertions (frameshifts). The sensitivity of these strains, engineered specifically for mutagen evaluation, has been genetically enhanced by eliminating their Excision Repair system. In addition, they lack the lipopolysaccharide coat that normally surrounds Salmonella cells. The absence of this barrier facilitates the penetration of potential mutagens into The Cell. Another crucial feature of this mutagen detection system is The addition of a mammalian Liver homogenate. Recall that some potential carcinogens are converted into their active forms by the enzymatic systems of the liver or other mammalian Tissues (Section 20.21). Because bacteria lack these Enzymes, a few milligrams of liver homogenate are applied to the plate to activate such mutagens. For example, a compound with a reactive side chain (enabling it to form a covalent bond with DNA) and an aromatic group (allowing it to intercalate) exhibits significantly greater mutagenicity than a similar compound consisting of an aromatic group alone.

Today, the Salmonella test is widely used to assess the mutagenic and carcinogenic hazards of a vast array of chemical compounds. This rapid and inexpensive bacterial mutagenicity assay complements epidemiological surveys and animal testing, which are invariably more labor-intensive, slow, and costly. The Salmonella mutagenicity test originated from fundamental studies of Gene-protein relationships in bacteria. It is a striking example of how basic research in molecular biology can make a direct and vital contribution to public health.