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Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000.

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An Introduction to Genetic Analysis. 7th edition.

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Induced mutations

Mutational specificity

When we observe the distribution of mutations induced by different mutagens, we see a distinct specificity that is characteristic of each mutagen. Such mutational specificity was first noted in the phage T4 rII system by Benzer in 1961. Specificity arises from a given mutagen’s “preference” both for a certain type of mutation (for example, GC → AT transitions) and for certain mutational sites (hot spots). Figure 16-13 shows the mutational specificity in lacI of three mutagens described later: ethylmethanesulfonate (EMS), ultraviolet (UV) light, and aflatoxin B1 (AFB1). The graphs show the distribution of base-substitution mutations that create chain-terminating UAG codons. Figure 16-13 is similar to Figure 9-26, which shows the distribution of mutations in rII, except that the specific sequence changes are known for each lacI site, allowing the graphs to be broken down into each category of substitution.

Figure 16-13. Specificity of mutagens.

Figure 16-13

Specificity of mutagens. The distribution of mutations among 36 sites in the lacI gene is shown for three mutagens: EMS, UV light, and aflatoxin B1. The height of each bar represents the number of occurrences of mutations at the respective site. Some (more...)

Figure 16-13 reveals the two components of mutational specificity. First, each mutagen shown favors a specific category of substitution. For example, EMS and UV favor GC → AT transitions, whereas AFB1 favors GC → TA transversions. These preferences are related to the different mechanisms of mutagenesis. Second, even within the same category, there are large differences in mutation rate. These differences can be seen best with UV light for the GC → AT changes. Some aspect of the surrounding DNA sequence must cause these differences. In some cases, the cause of mutational hot spots can be determined by DNA sequence studies, as previously described for 5-methylcytosine residues and for certain frameshift sites (Figures 16-5 and 16-9). In many examples of mutagen-induced hot spots, the precise reason for the high mutability of specific sites is still unknown. However, high lesion frequency at some sites and reduced repair at certain sites are sometimes causes of hot spots.

Mechanisms of mutagenesis

Mutagens induce mutations by at least three different mechanisms. They can replace a base in the DNA, alter a base so that it specifically mispairs with another base, or damage a base so that it can no longer pair with any base under normal conditions.

Incorporation of base analogs.  

Some chemical compounds are sufficiently similar to the normal nitrogen bases of DNA that they occasionally are incorporated into DNA in place of normal bases; such compounds are called base analogs. Once in place, these analogs have pairing properties unlike those of the normal bases; thus, they can produce mutations by causing incorrect nucleotides to be inserted opposite them in replication. The original base analog exists in only a single strand, but it can cause a nucleotide-pair substitution that is replicated in all DNA copies descended from the original strand.

For example, 5-bromouracil (5-BU) is an analog of thymine that has bromine at the C-5 position in place of the CH3 group found in thymine. This change does not affect the atoms that take part in hydrogen bonding in base pairing, but the presence of the bromine significantly alters the distribution of electrons in the base. The normal structure (the keto form) of 5-BU pairs with adenine, as shown in Figure 16-14a. 5-BU can frequently change to either the enol form or an ionized form; the latter pairs in vivo with guanine (Figure 16-14b). Thus, the nature of the pair formed in replication will depend on the form of 5-BU at the moment of pairing (Figure 16-15). 5-BU causes transitions almost exclusively, as predicted in Figures 16-14 and 16-15.

Figure 16-14. Alternative pairing possibilities for 5-bromouracil (5-BU).

Figure 16-14

Alternative pairing possibilities for 5-bromouracil (5-BU). 5-BU is an analog of thymine that can be mistakenly incorporated into DNA as a base. It has a bromine atom in place of the methyl group. (a) In its normal keto state, 5-BU mimics the pairing behavior (more...)

Figure 16-15. The mechanism of 5-BU mutagenesis.

Figure 16-15

The mechanism of 5-BU mutagenesis. 5-BU causes mutations when it is incorporated in one form and then shifts to another form. (a) In its normal keto state, 5-BU pairs like thymine (5-BUT). Thus, 5-BU is incorporated across from adenine and subsequently (more...)

Another analog widely used in research is 2-amino-purine (2-AP), which is an analog of adenine that can pair with thymine but can also mispair with cytosine when protonated, as shown in Figure 16-16. Therefore, when 2-AP is incorporated into DNA by pairing with thymine, it can generate AT → GC transitions by mispairing with cytosine in subsequent replications. Or, if 2-AP is incorporated by mispairing with cytosine, then GC → AT transitions will result when it pairs with thymine. Genetic studies have shown that 2-AP, like 5-BU, is very specific for transitions.

Figure 16-16. Alternative pairing possibilities for 2-aminopurine (2-AP), an analog of adenine.

Figure 16-16

Alternative pairing possibilities for 2-aminopurine (2-AP), an analog of adenine. Normally, 2-AP pairs with thymine (a), but in its protonated state it can pair with cytosine (b).

Specific mispairing.  

Some mutagens are not incorporated into the DNA but instead alter a base, causing specific mispairing. Certain alkylating agents, such as ethylmethanesulfonate (EMS) and the widely used nitrosoguanidine (NG), operate by this pathway:

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Although such agents add alkyl groups (an ethyl group in EMS and a methyl group in NG) to many positions on all four bases, mutagenicity is best correlated with an addition to the oxygen at the 6 position of guanine to create an O-6-alkylguanine. This addition leads to direct mispairing with thymine, as shown in Figure 16-17, and would result in GC → AT transitions at the next round of replication. As expected, determinations of mutagenic specificity for EMS and NG show a strong preference for GC → AT transitions (see the data for EMS shown in Figure 16-13). Alkylating agents can also modify the bases in dNTPs (where N is any base), which are precursors in DNA synthesis.

Figure 16-17. Alkylation-induced specific mispairing.

Figure 16-17

Alkylation-induced specific mispairing. The alkylation (in this case, EMS-generated ethylation) of the O-6 position of guanine and the O-4 position of thymine can lead to direct mispairing with thymine and guanine, respectively, as shown here. In bacteria, (more...)

The intercalating agents form another important class of DNA modifiers. This group of compounds includes proflavin, acridine orange, and a class of chemicals termed ICR compounds (Figure 16-18a). These agents are planar molecules, which mimic base pairs and are able to slip themselves in (intercalate) between the stacked nitrogen bases at the core of the DNA double helix (Figure 16-18b). In this intercalated position, the agent can cause single-nucleotide-pair insertions or deletions. Intercalating agents may also stack between bases in single-stranded DNA; in so doing, they may stabilize bases that are looped out during frameshift formation, as depicted in the Streisinger model (Figure 16-4).

Figure 16-18. Intercalating agents.

Figure 16-18

Intercalating agents. (a) Structures of the common agents proflavin, acridine orange, and ICR-191. (b) An intercalating agent slips between the nitrogenous bases stacked at the center of the DNA molecule. This occurrence can lead to single-nucleotide-pair (more...)

Base damage.  

A large number of mutagens damage one or more bases, so no specific base pairing is possible. The result is a replication block, because DNA synthesis will not proceed past a base that cannot specify its complementary partner by hydrogen bonding. In bacterial cells, such replication blocks can be bypassed by inserting nonspecific bases. The process requires the activation of a special system, the SOS system (Figure 16-19). The name SOS comes from the idea that this system is induced as an emergency response to prevent cell death in the presence of significant DNA damage. SOS induction is a last resort, allowing the cell to trade death for a certain level of mutagenesis.

Figure 16-19. The SOS system.

Figure 16-19

The SOS system. DNA polymerase III, shown in blue, stops at a noncoding lesion, such as the T–C photodimer shown here, generating single-stranded regions that attract the Ssb protein (dark purple) and RecA (light purple), which forms filaments. The (more...)

Exactly how the SOS bypass system functions is not clear, although in E. coli it is known to be dependent on at least three genes, recA (which also has a role in general recombination), umuC, and umuD. Current models for SOS bypass suggest that the UmuC and UmuD proteins combine with the polymerase III DNA replication complex to loosen its otherwise strict specificity and permit replication past noncoding lesions.

Figure 16-19 shows a model for the bypass system operating after DNA polymerase III stalls at a type of damage called a TC photodimer. Because replication can restart downstream from the dimer, a single-stranded region of DNA is generated. This region attracts the stabilizing protein, called single-strand-binding protein (Ssb), as well as the RecA protein, which forms filaments and signals the cell to synthesize the UmuC and UmuD proteins. The UmuD protein binds to the filaments and is cleaved by the RecA protein to yield a shortened version termed UmuD′, which then recruits the UmuC protein to form a complex that allows DNA polymerization to continue past the dimer, adding bases across from the dimer with a high error frequency (see Figure 16-19).

Therefore mutagens that damage specific base-pairing sites are dependent on the SOS system for their action. The category of SOS-dependent mutagens is important, because it includes most cancer-causing agents (carcinogens), such as ultraviolet light, aflatoxin B1, and benzo(a)pyrene (discussed later).

How does the SOS system take part in the recovery of mutations after mutagenesis? Does the SOS system lower the fidelity of DNA replications so much (to permit the bypass of noncoding lesions) that many replication errors occur, even for undamaged DNA? If this hypothesis were correct, most mutations generated by different SOS-dependent mutagens would be similar, rather than specific to each mutagen. Most mutations would result from the action of the SOS system itself on undamaged DNA. The mutagen, then, would play the indirect role of inducing the SOS system. Studies of mutational specificity, however, have shown that this is not the case. Instead, a series of different SOS-dependent mutagens have markedly different specificities, as seen for UV light and aflatoxin B1 in Figure 16-13. Each mutagen induces a unique distribution of mutations. Therefore, the mutations must be generated in response to specific damaged base pairs. The type of lesion differs in many cases. Some of the most widely studied lesions include UV photoproducts and apurinic sites.

Ultraviolet light generates a number of photoproducts in DNA. Two different lesions that occur at adjacent pyrimidine residues—the cyclobutane pyrimidine photodimer and the 6-4 photoproduct (Figure 16-20)—have been most strongly correlated with mutagenesis. These lesions interfere with normal base pairing; hence, induction of the SOS system is required for mutagenesis. The insertion of incorrect bases across from UV photoproducts is at the 3′ position of the dimer, and more frequently for 5′-CC-3′ and 5′-TC-3′ dimers. The C → T transition is the most frequent mutation, but other base substitutions (transversions) and frameshifts also are stimulated by UV light, as are duplications and deletions. The mutagenic specificity of UV light is illustrated in Figure 16-13.

Figure 16-20. (a) Structure of a cyclobutane pyrimidine dimer.

Figure 16-20

(a) Structure of a cyclobutane pyrimidine dimer. Ultraviolet light stimulates the formation of a four-membered cyclobutane ring (green) between two adjacent pyrimidines on the same DNA strand by acting on the 5,6 double bonds. (b) Structure of the 6-4 (more...)

Ionizing radiation

Ionizing radiation results in the formation of ionized and excited molecules that can cause damage to cellular components and to DNA. Because of the aqueous nature of biological systems, the molecules generated by the effects of ionizing radiation on water produce the most damage. Many different types of reactive oxygen specials are produced, including superoxide radicals, such as ·OH. The most biologically relevant reaction products are ·OH, O2 , and H2O2. These species can damage bases and cause different adducts and degradation products. Among the most prevalent, which result in mutations, are thymine glycol and 8-oxodG, pictured in Figure 16-10. Ionizing radiation can cause breakage of the N-glycosydic bond, leading to the formation of AP sites, and can cause strand breaks that are responsible for most of the lethal effects of such radiation.

Aflatoxin B1 is a powerful carcinogen. It generates apurinic sites following the formation of an addition product at the N-7 position of guanine (Figure 16-21). Studies with apurinic sites generated in vitro demonstrated a requirement for the SOS system and showed that the SOS bypass of these sites leads to the preferential insertion of an adenine across from an apurinic site. Thus agents that cause depurination at guanine residues should preferentially induce GC → TA transversions. Can you see why the insertion of an adenine across from an apurinic site derived from a guanine would generate this substitution at the next round of replication? Figure 16-13 shows the genetic analysis of many base substitutions induced by AFB1. You can verify that most of the substitutions are indeed GC → TA transversions.

Figure 16-21. The binding of metabolically activated aflatoxin B1 to DNA.

Figure 16-21

The binding of metabolically activated aflatoxin B1 to DNA.

AFB1 is a member of a class of chemical carcinogens known as bulky addition products when they bind covalently to DNA. Other examples include the diol epoxides of benzo(a)pyrene, a compound produced by internal combustion engines. For many different compounds, it is not yet clear which DNA addition products play the principal role in mutagenesis. In some cases, the mutagenic specificity suggests that depurination may be an intermediate step in mutagenesis; in others, the question of which mechanism is operating is completely open.

MESSAGE

Mutagens induce mutations by a variety of mechanisms. Some mutagens mimic normal bases and are incorporated into DNA, where they can mispair. Others damage bases and either cause specific mispairing or destroy pairing by causing nonrecognition of bases. In the latter case, a bypass system, the SOS system, must be induced to allow replication past the lesion.

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By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21936

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