An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
Chapter 16
Mechanisms of Gene Mutation
Key Concepts
Mutations can occur spontaneously owing to several different mechanisms, including errors of
DNA replication and spontaneous damage to the DNA.
Mutagens are agents that increase the frequency of mutagenesis, usually by altering the DNA.
Potentially mutagenic and carcinogenic compounds can be detected easily by mutagenesis
tests with bacterial systems.
Biological repair systems eliminate many potentially mutagenic alterations in the DNA.
Cells lacking certain repair systems have higher than normal mutation rates.
Introduction
This chapter describes the main types of molecular processes that give rise to the mutant
alleles discussed in the previous chapter. Such processes are relevant not only to experimental
genetics but also have direct bearing on human health.
Molecular basis of gene mutations
Gene mutations can arise spontaneously or they can be induced. Spontaneous mutations are
naturally occurring mutations and arise in all cells. Induced mutations are produced when an
organism is exposed to a mutagenic agent, or mutagen; such mutations typically occur at much
higher frequencies than spontaneous mutations do.
To understand the mechanisms of gene mutation requires analysis at the level of DNA and
protein molecules. Preceding chapters have described models for DNA and protein structure
and have considered the nature of mutations that alter these structures (see, for instance, Table
15-1).
Molecular genetics techniques can be used to determine the sequence of large segments of
DNA and the sequence changes resulting from mutations. Sequencing has greatly increased
our understanding of the pathways that lead to mutagenesis and has even helped to unravel the
mysteries of mutational hot spots—genetic sites with a penchant for mutating.
Much work on the molecular basis of mutation has been carried out in single-celled bacteria
and their viruses. However, many mutations leading to inherited diseases in humans also have
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
been analyzed. We shall review some of the findings of these studies. We shall also consider
biological repair mechanisms, because repair systems play a key role in mutagenesis,
operating to lower the final observed mutation rates. For example, in Escherichia coli, with all
repair systems functioning, base substitutions occur at rates of 10−10 to 10−9 per base pair per
cell per generation. As a general principle, base substitutions arise by a perturbation of the
normal pairing of complementary bases.
Spontaneous mutations
Spontaneous mutations arise from a variety of sources, including errors in DNA replication,
spontaneous lesions, and transposable genetic elements. The first two are considered in this
section; the third is examined in Chapter 20.
Errors in DNA replication
An error in DNA replication can occur when an illegitimate nucleotide pair (say, A–C) forms
in DNA synthesis, leading to a base substitution.
Each of the bases in DNA can appear in one of several forms, called tautomers, which are
isomers that differ in the positions of their atoms and in the bonds between the atoms. The
forms are in equilibrium. The keto form of each base is normally present in DNA (Figure
16-1), whereas the imino and enol forms of the bases are rare. The ability of the wrong
tautomer of one of the standard bases to mispair and cause a mutation in the course of DNA
replication was first noted by Watson and Crick when they formulated their model for the
structure of DNA (Chapter 8). Figure 16-2 demonstrates some possible mispairs resulting from
the change of one tautomer into another, termed a tautomeric shift.
Mispairs can also result when one of the bases becomes ionized. This type of mispair may
occur more frequently than mispairs due to imino and enol forms of bases.
Transitions.
All the mispairs described so far lead to transition mutations, in which a purine substitutes for
a purine or a pyrimidine for a pyrimidine (Figure 16-3). The bacterial DNA polymerase III
(Chapter 8) has an editing capacity that recognizes such mismatches and excises them, thus
greatly reducing the observed mutations. Another repair system (described later in this chapter)
corrects many of the mismatched bases that escape correction by the polymerase editing
function.
Transversions.
In transversion mutations, a pyrimidine substitutes for a purine or vice versa. Transversions
cannot be generated by the mismatches depicted in Figure 16-2. With bases in the DNA in the
normal orientation, creation of a transversion by a replication error would require, at some
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
point in the course of replication, mispairing of a purine with a purine or a pyrimidine with a
pyrimidine. Although the dimensions of the DNA double helix render such mispairs
energetically unfavorable, we now know from X-ray diffraction studies that G–A pairs, as well
as other purine–purine pairs, can form.
Frameshift mutations.
Replication errors can also lead to frameshift mutations. Recall from Chapter 10 that such
mutations result in greatly altered proteins.
In the mid-1960s, George Streisinger and his coworkers deduced the nucleotide sequence
surrounding different sites of frameshift mutations in the lysozyme gene of phage T4. They
found that these mutations often occurred at repeated sequences and formulated a model to
account for frameshifts in DNA synthesis. In the Streisinger model (Figure 16-4), frameshifts
arise when loops in single-stranded regions are stabilized by the “slipped mispairing” of
repeated sequences.
In the 1970s, Jeffrey Miller and his co-workers examined mutational hot spots in the lacI gene
of E. coli. As already mentioned, hot spots are sites in a gene that are much more mutable than
other sites. The lacI work showed that certain hot spots result from repeated sequences, just as
predicted by the Streisinger model. Figure 16-5 depicts the distribution of spontaneous
mutations in the lacI gene. Compare this distribution with that in the rII genes of T4 seen by
Benzer (see Figure 9-26). Note how one or two mutational sites dominate the distribution in
both cases. In lacI, a four-base-pair sequence repeated three times in tandem in the wild type is
the cause of the hot spots (for simplicity, only one strand of the double strand of DNA is
indicated):
The major hot spot, represented here by the mutations FS5, FS25, FS45, and FS65, results
from the addition of one extra set of the four bases CTGG to one strand of the DNA. This hot
spot reverts at a high rate, losing the extra set of four bases. The minor hot spot, represented
here by the mutations FS2 and FS84, results from the loss of one set of the four bases CTGG.
This mutant does not readily regain the lost set of four base pairs.
How can the Streisinger model explain these observations? The model predicts that the
frequency of a particular frameshift depends on the number of base pairs that can form during
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
the slipped mispairing of repeated sequences. The wild-type sequence shown for the lacI gene
can slip out one CTGG sequence and stabilize structure by forming nine base pairs. (Can you
work this out by applying the model in Figure 16-4 to the sequence shown for lacI?) Whether
a deletion or an addition is generated depends on whether the slippage occurs on the template
or on the newly synthesized strand, respectively. In a similar fashion, the addition mutant can
slip out one CTGG sequence and stabilize a structure with 13 base pairs (verify this for the
FS5 sequence shown for lacI), which explains the rapid reversion of mutations such as FS5.
However, there are only five base pairs available to stabilize a slipped-out CTGG in the
deletion mutant, accounting for the infrequent reversion of mutations such as FS2 in the
sequence shown for lacI.
Deletions and duplications.
Large deletions (more than a few base pairs) constitute a sizable fraction of sponta-neous
mutations, as shown in Figure 16-5. The majority, although not all, of the deletions occur at
repeated sequences. Figure 16-6 shows the results for the first 12 deletions analyzed at the
DNA sequence level, presented by Miller and his co-workers in 1978. Further studies showed
that hot spots for deletions are in the longest repeated sequences. Duplications of segments of
DNA have been observed in many organisms. Like deletions, they often occur at sequence
repeats.
How do deletions and duplications form? Several mechanisms could account for their
formation. Deletions may be generated as replication errors. For example, an extension of the
Streisinger model of slipped mispairing (Figure 16-4) could explain why deletions
predominate at short repeated sequences. Alternatively, deletions and duplications could be
generated by recombinational mechanisms (to be described in Chapter 19).
Spontaneous lesions
In addition to replication errors, spontaneous lesions, naturally occurring damage to the DNA,
can generate mutations. Two of the most frequent spontaneous lesions result from
depurination and deamination.
Depurination, the more common of the two, consists of the interruption of the glycosidic bond
between the base and deoxyribose and the subsequent loss of a guanine or an adenine residue
from the DNA (Figure 16-7). A mammalian cell spontaneously loses about 10,000 purines
from its DNA in a 20-hour cell-generation period at 37°C. If these lesions were to persist, they
would result in significant genetic damage because, in replication, the resulting apurinic sites
cannot specify a base complementary to the original purine. However, as we shall see later in
the chapter, efficient repair systems remove apurinic sites. Under certain conditions (to be
described later), a base can be inserted across from an apurinic site; this insertion will
frequently result in a mutation.
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Chapter 16
Mechanisms of Gene Mutation
The deamination of cytosine yields uracil (Figure 16-8a). Unrepaired uracil residues will pair
with adenine in replication, resulting in the conversion of a G–C pair into an A–T pair (a GC
→ AT transition). In 1978, deaminations at certain cytosine residues were found to be the
cause of one type of mutational hot spot. DNA sequence analysis of GC → AT transition hot
spots in the lacI gene showed that 5-methylcytosine residues are pres-ent at each hot spot.
(Certain bases in prokaryotes and eukaryotes are methylated.) Some of the data from this lacI
study are shown in Figure 16-9. The height of each bar on the graph represents the frequency
of mutations at each of a number of sites. It can be seen that the positions of 5-methylcytosine
residues correlate nicely with the most mutable sites.
Why are 5-methylcytosines hot spots for mutations? One of the repair enzymes in the cell,
uracil-DNA glycosylase, recognizes the uracil residues in the DNA that arise from
deaminations and excises them, leaving a gap that is subsequently filled (a process to be
described later in the chapter). However, the deamination of 5-methylcytosine (Figure 16-8b)
generates thymine (5-methyluracil), which is not recognized by the enzyme uracil-DNA
glycosylase and thus is not repaired. Therefore, C → T transitions generated by deamination
are seen more frequently at 5-methylcytosine sites, because they escape this repair system.
A consequence of the frequent mutation of 5-methylcytosine to thymine is the
underrepresentation of CpG dinucleotides in higher cells, because this sequence is methylated
to give 5-methyl-CpG, which is gradually converted into TpG.
Oxidatively damaged bases represent a third type of spontaneous lesion implicated in
mutagenesis. Active oxygen species, such as superoxide radicals (O2·), hydrogen peroxide
(H2O2), and hydroxyl radicals (OH·), are produced as by-products of normal aerobic
metabolism. They can cause oxidative damage to DNA, as well as to precursors of DNA (such
as GTP), which results in mutation and which has been implicated in a number of human
diseases. Figure 16-10 shows two products of oxidative damage. The
8-oxo-7-hydrodeoxyguanosine (8-oxodG, or GO) product frequently mispairs with A,
resulting in a high level of G → T transversions. Thymidine glycol blocks DNA replication if
unrepaired but has not yet been implicated in mutagenesis.
MESSAGE
Spontaneous mutations can be generated by different processes. Replication errors
and spontaneous lesions generate most of the base-substitution and frameshift
mutations. Replication errors may also cause some deletions that occur in the absence
of mutagenic treatment.
Spontaneous mutations and human diseases
DNA sequence analysis has revealed the mutations responsible for a number of human
hereditary diseases. The previously discussed studies of bacterial mutations allow us to
suggest mechanisms that cause these human disorders.
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
A number of these disorders are due to deletions or duplications involving repeated sequences.
For example, mitochondrial encephalomyopathies are a group of disorders affecting the
central nervous system or the muscles (Kearns-Sayre syndrome). They are characterized by
dysfunction of oxidation phosphorylation (a function of the mitochondria) and by changes in
mitochondrial structure. These disorders have been shown to result from deletions that occur
between repeated sequences. Figure 16-11 depicts one of these deletions. Note how similar it
is in form to the spontaneous E. coli deletions shown in Figure 16-6.
A common mechanism that is responsible for a number of genetic diseases is the expansion of
a three-base-pair repeat, as in fragile X syndrome (Figure 16-12). This syndrome is the most
common form of inherited mental retardation, occurring in close to 1 of 1500 males and 1 of
2500 females. It is evidenced cytologically by a fragile site in the X chromosome that results
in a break in vitro.
The inheritance of fragile X syndrome is unusual in that 20 percent of the males with a fragile
X chromosome are phenotypically normal but transmit the affected chromosome to their
daughters, who also appear normal. These males are said to be normally transmitting males
(NTMs). However, the sons of the daughters of the NTMs frequently display symptoms. The
fragile X syndrome results from mutations in a (CGG) n repeat in the coding sequence of the
FMR-1 gene. Patients with the disease show specific methylation, induced by the mutation, at
a nearby CpG cluster, resulting in reduced FMR-1 expression.
Why do symptoms develop in some persons with a fragile X chromosome and not in others?
The answer seems to lie in the number of CGG repeats in the FMR-1 gene. Humans normally
show a considerable variation in the number of CGG repeats in the FMR-1 gene, ranging from
6 to 54, with 29 repeats in the most frequent allele. [The variation in CGG repeats produces a
corresponding variation in the number of arginine residues (CGG is an arginine codon) in the
FMR-1-encoded protein.] Both NTMs and their daughters have a much larger number of
repeats, ranging from 50 to 200. These increased repeats have been termed premutations. All
premutation alleles are unstable. The males and females with symptoms of the disease, as well
as many carrier females, have additional insertions of DNA, suggesting repeat numbers of 200
to 1300. The frequency of expansion has been shown to increase with the size of the DNA
insertion (and thus, presumably, with the number of repeats). Apparently, the number of
repeats in the premutation alleles found in NTMs and their daughters is above a certain
threshold and thus is much more likely to expand to a full mutation than is the case for normal
persons.
The proposed mechanism for these repeats is a slipped mispairing in DNA synthesis (as shown
in Figure 16-6) involving a one-step expansion of the four-base-pair sequence CTGG.
However, the extraordinarily high frequency of mutation at the three-base-pair repeats in the
fragile X syndrome suggests that in human cells, after a threshold level of about 50 repeats,
the replication machinery cannot faithfully replicate the correct sequence, and large variations
in repeat numbers result.
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
A second inherited disease, X-linked spinal and bulbar muscular atrophy (known as Kennedy
disease), also results from the amplification of a three-base-pair repeat, in this case a repeat of
the CAG triplet. Kennedy disease, which is characterized by progressive muscle weakness and
atrophy, results from mutations in the gene that encodes the androgen receptor. Normal
persons have an average of 21 CAG repeats in this gene, whereas affected patients have
repeats ranging from 40 to 52.
Myotonic dystrophy, the most common form of adult muscular dystrophy, is yet another
example of sequence expansion causing a human disease. Susceptible families display an
increase in severity of the disease in successive generations; this increase is caused by the
progressive amplification of a CTG triplet at the 3′ end of a transcript. Normal people possess,
on average, five copies of the CTG repeat; mildly affected people have approximately 50
copies, and severely affected people have more than 1000 repeats of the CTG triplet.
Additional examples of triplet expansion are still appearing—for instance, Huntington disease.
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 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
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Chapter 16
Mechanisms of Gene Mutation
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.
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.
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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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
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.
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).
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.
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.
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
Figure 16-19 shows a model for the bypass system operating after DNA polymerase III stalls
at a type of damage called a T–C 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.
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
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
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.
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.
Reversion analysis
Testing for the reversion of a mutation can tell us something about the nature of the mutation
or the action of a mutagen. For example, if a mutation cannot be reverted by action of the
mutagen that induced it, then the mutagen must have some relatively specific unidirectional
action. In a mutation induced by hydroxylamine (HA), for instance, it would be reasonable to
expect that the original mutation is GC → AT, which cannot be reverted by another specific
GC → AT event. Similarly, mutations that can be reverted by proflavin are in all likelihood
frameshift mutations; thus mutations induced by nitrous acid (NA), which are transitions,
should not be revertible by proflavin.
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
Transversions cannot be induced by the aforementioned agents, but they are definitely known
to be common among spontaneous mutations, as shown by studies of DNA and protein
sequencing. Thus, in the reversion test, if a mutation reverts spontaneously but does not revert
in response to a transition mutagen or a frameshift mutagen, then, by elimination, it is
probably a transversion.
Table 16-1 summarizes some reversion expectations based on simple assumptions from
reversion analysis. Recall that mutagen specificities depend on the organism, the genotype, the
gene studied, and the action of biological repair systems. Note that the kinds of logic
employed in the reversion test rely heavily on the assumption that the reversion events are not
due to suppressors or transposable elements; either of them would make inference from
reversion more difficult.
Relation between mutagens and carcinogens
Mutagenicity and carcinogenicity are clearly correlated. One study showed that 157 of 175
known carcinogens (approximately 90 percent) are also mutagens. The somatic mutation
theory of cancer holds that these agents cause cancer by inducing the mutation of somatic cells.
Thus, understanding mutagenesis is of great relevance to our society.
Induced mutations and human cancer
Understanding the specificity of mutagens in bacteria has led to the direct implication of
certain environmental mutagens in the causation of human cancers. Ultraviolet light and
aflatoxin B1 have long been suspected of causing skin cancer and liver cancer, respectively.
Now, DNA sequence analysis of mutations in a human cancer gene has provided direct
evidence of their involvement. The gene in question is termed p53 and is one of a number of
tumor-suppressor genes—genes that encode proteins that suppress tumor formation. (We will
learn more about these genes in Chapter 23.) A sizable proportion of human cancer patients
have mutated tumor-suppressor genes.
Liver cancer is prevalent in southern Africa and East Asia, and a high exposure to AFB1 in
these regions has been correlated with the high incidence of liver cancer. When p53 mutations
in cancer patients were analyzed, G → T transversions, the signature of AFB1-induced
mutations, were found in liver cancer patients from South Africa and East Asia but not in
patients from these regions with lung, colon, or breast cancer. On the other hand, p53
mutations in liver cancer patients from areas of low AFB1 exposure did not result from G → T
transversions. These findings, together with the results from the mutagenic specificity studies
of AFB1 (see Figure 16-13), allow us to conclude that AFB1-induced mutations are a prime
cause of liver cancer in South Africa and East Asia.
Sequencing p53 mutations has also strengthened the link between UV and human skin cancers.
The majority of invasive human squamous cell carcinomas analyzed so far have p53 mutations,
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Chapter 16
Mechanisms of Gene Mutation
all of them mutations at dipyrimidine sites, most of which are C → T substitutions when the C
is the 3′ pyrimidine of a TC dimer. This is the profile of UV-induced mutations. In addition,
several tumors have p53 mutations resulting from a CC → TT double base change, which is
found most frequently among UV-induced mutations.
The modern environment exposes everyone to a wide variety of chemicals in drugs, cosmetics,
food preservatives, pesticides, compounds used in industry, pollutants, and so forth. Many of
these compounds have been shown to be carcinogenic and mutagenic. Examples include the
food preservative AF-2, the food fumigant ethylene dibromide, the antischistosome drug
hycanthone, several hair-dye additives, and the industrial compound vinyl chloride; all are
potent, and some have subsequently been subjected to government control. However,
hundreds of new chemicals and products appear on the market each week. How can such vast
numbers of new agents be tested for carcinogenicity before much of the population has been
exposed to them?
Ames test
Many test systems have been devised to screen for carcinogenicity. These tests are time
consuming, typically requiring laborious research with small mammals. More rapid tests do
exist that make use of microbes (such as fungi or bacteria) and test for mutagenicity rather
than carcinogenicity. The most widely used test was developed in the 1970s by Bruce Ames,
who worked with Salmonella typhimurium. This Ames test uses two auxotrophic histidine
mutations, which revert by different molecular mechanisms (Figure 16-22). Further properties
were genetically engineered into these strains to make them suitable for mutagen detection.
First, they carry a mutation that inactivates the excision-repair system (described later).
Second, they carry a mutation that eliminates the protective lipopolysaccharide coating of
wild-type Salmonella to facilitate the entry of many different chemicals into the cell.
Bacteria are evolutionarily a long way removed from humans. Can the results of a test on
bacteria have any real significance in detecting chemicals that are dangerous for humans? First,
we have seen that the genetic and chemical nature of DNA is identical in all organisms, so a
compound acting as a mutagen in one organism is likely to have some mutagenic effects in
other organisms. Second, Ames devised a way to simulate the human metabolism in the
bacterial system. In mammals, much of the important processing of ingested chemicals takes
place in the liver, where externally derived compounds normally are detoxified or broken
down. In some cases, the action of liver enzymes can create a toxic or mutagenic compound
from a substance that was not originally dangerous (Figure 16-23). Ames incorporated
mammalian liver enzymes in his bacterial test system, using rat livers for this purpose. Figure
16-24 outlines the procedure used in the Ames test.
Chemicals detected by this test can be regarded not only as potential carcinogens (sources of
somatic mutations), but also as possible causes of mutations in germinal cells. Because the test
system is so simple and inexpensive, many laboratories throughout the world now routinely
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Chapter 16
Mechanisms of Gene Mutation
test large numbers of potentially hazardous compounds for mutagenicity and potential
carcinogenicity.
Biological repair mechanisms
As we have seen in the preceding discussions, there are many potential threats to the fidelity of
DNA replication. Not only is there an inherent error rate for the replication of DNA, but there
are also spontaneous lesions that can provoke additional errors. Moreover, mutagens in the
environment can damage DNA and greatly increase the mutation rate.
Living cells have evolved a series of enzymatic systems that repair DNA damage in a variety
of ways. Failure of these systems can lead to a higher mutation rate. A number of human
diseases including certain types of cancer can be attributed to defects in DNA repair, as we
shall see later. Let's first examine some of the characterized repair pathways and then consider
how the cell integrates these systems into an overall strategy for repair.
We can divide repair pathways into several categories.
Prevention of errors before they happen
Some enzymatic systems neutralize potentially damaging compounds before they even react
with DNA. One example of such a system is the detoxification of superoxide radicals
produced during oxidative damage to DNA: the enzyme superoxide dismutase catalyzes the
conversion of the superoxide radicals into hydrogen peroxide, and the enzyme catalase, in turn,
converts the hydrogen peroxide into water. Another error-prevention pathway depends on the
protein product of the mutT gene: this enzyme prevents the incorporation of 8-oxodG (see
Figure 16-10), which arises by oxidation of dGTP, into DNA by hydrolyzing the triphosphate
of 8-oxodG back to the monophosphate.
Direct reversal of damage
The most straightforward way to repair a lesion, once it occurs, is to reverse it directly, thereby
regenerating the normal base. Reversal is not always possible, because some types of damage
are essentially irreversible. In a few cases, however, lesions can be repaired in this way. One
case is a mutagenic photodimer caused by UV light (see Figure 16-20). The cyclobutane
pyrimidine photodimer can be repaired by a photolyase that has been found in bacteria and
lower eukaryotes but not in humans. The enzyme binds to the photodimer and splits it, in the
presence of certain wavelengths of visible light, to generate the original bases (Figure 16-25).
This enzyme cannot operate in the dark, so other repair pathways are required to remove UV
damage. A photolyase that reverses the 6-4 photoproducts has been detected in plants and
Drosophila.
Alkyltransferases also are enzymes taking part in the direct reversal of lesions. They remove
certain alkyl groups that have been added to the O-6 positions of guanine (Figure 16-17) by
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
such agents as NG and EMS. The methyltransferase from E. coli has been well studied. This
enzyme transfers the methyl group from O-6-methylguanine to a cysteine residue on the
protein. When this happens, the enzyme is inactivated, so this repair system can be saturated if
the level of alkylation is high enough.
Excision-repair pathways
General excision repair.
Also termed nucleotide excision repair, this system includes the breaking of a phosphodiester
bond on either side of the lesion, on the same strand, resulting in the excision of an
oligonucleotide. This excision leaves a gap that is filled by repair synthesis, and a ligase seals
the breaks. In prokaryotes, 12 or 13 nucleotides are removed; whereas, in eukaryotes, from 27
to 29 nucleotides are eliminated. Figure 16-26 depicts the incision pattern in each case.
In E. coli, the products of the uvrA, B, and C genes constitute the excinuclease. The UvrA
protein, which recognizes the damaged DNA, forms a complex with UvrB and leads the UvrB
subunit to the damage site before dissociating. The UvrC protein then binds to UvrB. Each of
these subunits makes an incision. The short DNA 12-mer is unwound and released by another
protein, helicase II. Figure 16-27 shows a detailed view of these excision events.
The human excinuclease is considerably more complex than its bacterial counterpart and
includes at least 17 proteins. However, the basic steps are the same as those in E. coli.
Coupling of transcription and repair.
The involvement of TFIIH, a transcription factor, in excision repair underscores the fact that
transcription and repair are coupled. In both eukaryotes and prokaryotes, there is a preferential
repair of the transcribed strand of DNA for actively expressed genes. Figure 16-28 portrays a
mechanism for this coupling.
Specific excision pathways.
Certain lesions are too subtle to cause a distortion large enough to be recognized by the
uvrABC-encoded general excision-repair system and its counterparts in higher cells. Thus,
additional excision pathways are necessary.
DNA glycosylase repair pathway (base-excision repair).
DNA glycosylases do not cleave
phosphodiester bonds, but instead cleave N-glycosidic (base–sugar) bonds, liberating the
altered base and generating an apurinic or an apyrimidinic site, both called AP sites, because
they are biochemically equivalent (see Figure 16-7). This initial step is shown in Figure 16-29.
The resulting Ap site is then repaired by an AP endonuclease repair pathway (described in the
next subsection).
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
Numerous DNA glycosylases exist. One, uracil-DNA glycosylase, removes uracil from DNA.
Uracil residues, which result from the spontaneous deamination of cytosine (Figure 16-8), can
lead to a C → T transition if unrepaired. It is possible that the natural pairing partner of
adenine in DNA is thymine (5-methyluracil), rather than uracil, to allow the recognition and
excision of these uracil residues. If uracil were a normal constituent of DNA, such repair
would not be possible.
There is also a glycosylase that recognizes and excises hypoxanthine, the deamination product
of adenine. Other glycosylases remove alkylated bases (such as 3-methyladenine,
3-methylguanine, and 7-methylguanine), ring-opened purines, oxidatively damaged bases, and,
in some organisms, UV photodimers. New glycosylases are still being discovered.
AP endonuclease repair pathway.
All cells have endonucleases that attack the sites left
after the spontaneous loss of single purine or pyrimidine residues. The AP endonucleases are
vital to the cell, because, as noted earlier, spontaneous depurination is a relatively frequent
event. These enzymes introduce chain breaks by cleaving the phosphodiester bonds at AP sites.
This bond cleavage initiates an excision-repair process mediated by three further enzymes—an
exonuclease, DNA polymerase I, and DNA ligase (Figure 16-30).
Owing to the efficiency of the AP endonuclease repair pathway, it can be the final step of
other repair pathways. Thus, if damaged base pairs can be excised, leaving an AP site, the AP
endonucleases can complete the restoration to the wild type. This is what happens in the DNA
glycosylase repair pathway.
GO system.
Two glycosylases, the products of the mutM and mutY genes, work in concert
to prevent mutations arising from the 8-oxodG, or GO, lesion in DNA (see Figure 16-10).
Together with the product of the mutT gene mentioned earlier, these glycosylases form the GO
system. When GO lesions are generated in DNA by spontaneous oxidative damage, a
glycosylase encoded by mutM removes the lesion (Figure 16-31). Still, some GO lesions
persist and mispair with adenine. A second glycosylase, the product of the mutY gene,
removes the adenine from this specific mispair, leading to restoration of the correct cytosine
by repair synthesis (mediated by DNA polymerase I) and allowing subsequent removal of the
GO lesion by the mutM product.
The mutT product prevents incorporation of GO across from A. The human counterparts of the
mutT, mutY, and mutM gene products have been detected.
Postreplication repair
Mismatch repair.
Some repair pathways are capable of recognizing errors even after DNA replication has
already occurred. One such system, termed the mismatch repair system, can detect mismatches
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
that occur in DNA replication. Suppose you were to design an enzyme system that could
repair replication errors. What would this system have to be able to do? At least three things:
1. Recognize mismatched base pairs.
2. Determine which base in the mismatch is the incorrect one.
3. Excise the incorrect base and carry out repair synthesis.
The second point is the crucial property of such a system. Unless it is capable of
discriminating between the correct and the incorrect bases, the mismatch repair system could
not determine which base to excise. If, for example, a G–T mismatch occurs as a replication
error, how can the system determine whether G or T is incorrect? Both are normal bases in
DNA. But replication errors produce mismatches on the newly synthesized strand, so it is the
base on this strand that must be recognized and excised.
To distinguish the old, template strand from the newly synthesized strand, the mismatch repair
system in bacteria takes advantage of the normal delay in the postreplication methylation of
the sequence
The methylating enzyme is adenine methylase, which creates 6-methyladenine on each strand.
However, it takes the adenine methylase several minutes to recognize and modify the newly
synthesized GATC stretches. During that interval, the mismatch repair system can operate
because it can now distinguish the old strand from the new one by the methylation pattern.
Methylating the 6-position of adenine does not affect base pairing, and it provides a
convenient tag that can be detected by other enzyme systems. Figure 16-32 shows the
replication fork during mismatch correction. Note that only the old strand is methylated at
GATC sequences right after replication.
When the mismatched site has been identified, the mismatch repair system corrects the error.
Figure 16-33 depicts a model of how the mismatch repair system carries out the correction in
E. coli.
The mismatch repair system has also been characterized in humans. Two of the proteins,
hMSH2 and hMLH1, are very similar to their bacterial counterparts, MutS and MutL,
respectively. Figure 16-34 depicts how the hMSH2 protein, together with the G–T-binding
protein (GTBP), binds to the mismatches and then recruits the other components of the system,
hPMS2 and hMLH1, to effect repair of the mismatch.
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勇者并非无所畏惧，而是能判断出有比恐惧更重要的东西.
An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
Recombinational repair.
The recA gene, which has a role in SOS bypass (Figure 16-19), also takes part in
postreplication repair. Here the DNA replication system stalls at a UV photodimer or other
blocking lesion and then restarts past the block, leaving a single-stranded gap. In
recombinational repair, this gap is patched by DNA cut from the sister molecule (Figure
16-35a). This process seems to lead to few errors. SOS bypass, in contrast, is highly mutagenic,
as described earlier. Here the replication system continues past the lesion (Figures 16-19 and
16-35b), accepting noncomplementary nucleotides for new strand synthesis.
Strategy for repair
We can now assess the overall repair system strategy used by the cell. The many different
repair systems available to the cell are summarized in Table 16-2. It would be convenient if
enzymes could be used to directly reverse each specific lesion. However, sometimes that is not
chemically possible, and not every possible type of DNA damage can be anticipated.
Therefore, a general excision repair system is used to remove any type of damaged base that
causes a recognizable distortion in the double helix. When lesions are too subtle to cause such
a distortion, specific excision systems, glycosylases, or removal systems are designed. To
eliminate replication errors, a postreplication mismatch repair system operates; finally,
postreplication recombinational systems eliminate gaps across from blocking lesions that have
escaped the other repair systems.
A number of repair pathways are induced in response to damage, such as the SOS system (see
Figure 16-19), and many of the proteins participating in the repair of alkylation damage were
discussed previously.
MESSAGE
Repair enzymes play a crucial role in reducing genetic damage in living cells. The cell
has many different repair pathways at its command to eliminate potentially mutagenic
errors.
Mutators
As the preceding description of repair processes indicates, normal cells are programmed for
error prevention. The repair processes are so efficient that the observed basesubstitution rate is
as low as 10−10 to 10−9 per base pair per cell per generation in E. coli. However, mutant strains
with increased spontaneous mutation rates have been detected. Such strains are termed
mutators. In many cases, the mutator phenotype is due to a defective repair system. In humans,
these repair defects often lead to serious diseases.
In E. coli, the mutator loci mutH, mutL, mutU, and mutS affect components of the
postreplication mismatch repair system (see Figure 16-33), as does the dam locus, which
specifies the enzyme deoxyadenosine methylase. Strains that are Dam − cannot methylate
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
adenines at GATC sequences (see Figure 16-32), and so the mismatch repair system can no
longer discriminate between the template and the newly synthesized strands. This failure to
discriminate leads to a higher spontaneous mutation rate.
Mutations in the mutY locus result in GC → TA transversions, because many G–A mispairs
and all 8-oxodG–A mispairs are unrepaired (see the GO system described earlier). The mutM
gene encodes a glycosylase that removes 8-oxodG. Strains lacking mutM are mutators for the
GC → TA transversion. Strains that are MutT− have elevated rates of the AT → CG
transversion, because they lack an activity that prevents the incorporation of 8-oxodG across
from adenine.
Strains that are Ung− are missing the enzyme uracil DNA glycosylase. These mutants cannot
excise the uracil resulting from cytosine deaminations and, as a result, have elevated levels of
C → T transitions. The mutD locus is responsible for a very high rate of mutagenesis (at least
three orders of magnitude higher than normal). Mutations at this locus affect the proofreading
functions of DNA polymerase III (Chapter 8).
Repair defects and human diseases
Several human genetic diseases are known or suspected to be due to repair defects. These
defects often lead to an increased incidence of cancer. Table 16-3 summarizes information
about these diseases, which are usually autosomal recessive disorders. We consider two
examples.
Xeroderma pigmentosum (XP) results from a defect in any of the genes (complementation
groups) effecting nucleotide excision repair (see Figures 16-27 and 16-28). People suffering
from this disorder are extremely prone to UVinduced skin cancers (Figure 16-36) as a result of
exposure to sunlight and have frequent neurological abnormalities. The difference in UV
photosensitivity between normal and diseased cells is evident from the survival curves in
Figure 16-37.
Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common inherited
predispositions to cancer, affecting as many as 1 in 200 people in the Western world. Studies
have shown that this syndrome results from a loss of the mismatch repair system. Most
HNPCC results from a defect in genes that encode the human counterparts (and homologs) of
the bacterial MutS and MutL proteins (see Figure 16-34). The inheritance of HNPCC is
autosomal dominant. Cells with one functional copy of the mismatch repair genes have normal
mismatch repair activity, but tumor cell lines arise from cells that have lost the one functional
copy and are thus mismatch repair deficient. These cells display high mutation rates that
eventually result in tumor growth and proliferation.
Summary
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勇者并非无所畏惧，而是能判断出有比恐惧更重要的东西.
An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
Gene mutations can arise through many different processes. Spontaneous mutations can result
from replication errors or from spontaneous lesions, such as those generated by deamination or
depurination. (Recombination and transposable elements also can result in altered genes, as
described in Chapters 19 and 20.) Mutagens can increase the frequency of mutations. Some of
these agents act by mimicking a base and then mispairing in the course of DNA replication.
Others alter bases in the DNA and convert them into derivatives that mispair. A third class of
mutagens, which includes most carcinogens, damages DNA in such a way that replication is
blocked. The activation of an enzymatic pathway, termed the SOS system, is required to
replicate past the blocking lesions, resulting in mutations across from the blocking lesion.
Molecular studies of mutagenesis in bacteria can identify agents responsible for human
cancers. DNA sequencing carried out on mutations in the tumor-suppressor genes of patients
with liver or skin cancer has identified changes that closely resemble the characteristic
mutational changes caused by specific bacterial mutagens.
Our knowledge of the molecular basis of mutation can be exploited for useful purposes. One
example is the Ames test, which utilizes mutant bacterial strains to test compounds in the
environment for mutagenic activity. Owing to the correlation between mutagenicity and
carcinogenicity, the identification of potential carcinogens in the environment can be achieved
by this rapid assay.
Repair enzymes present in living cells greatly minimize genetic damage, thus preventing many
mutations. Mutant organisms lacking certain repair enzymes have higher than normal mutation
rates. In humans, repair deficiency leads to a variety of diseases and cancer susceptibilities.
Problems
1. The continuous sequence presented here (see the following page), which is not necessarily
translated, is from one of the two DNA strands in a genetic region. From the sequence, find
the mutations that would be expected to occur most frequently at the DNA level
spontaneously (without the aid of mutagens). Explain what each mutation is and why it
would be frequent.
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
(Problem 1 from Discovering Molecular Genetics by Jeffrey H. Miller. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1996.)
2. Describe the Streisinger model for frameshift formation. Show how this model can explain
mutational hot spots in the lacI gene of E. coli.
3. Diagram two different mechanisms for deletion formation. How do DNA-sequencing
experiments suggest these possibilities?
4. The bases in the DNA of Escherichia classicus are named after the musical keys A, B, C,
and E. Here, A pairs with E, and B pairs with C. Thus, there are A–E base pairs and B–C
base pairs in the double-stranded DNA.
You have the following eight sequences read 5′ to 3′. Here, just a single strand of DNA is
given; the second line is a continuation of the first line for each entry.
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An Introduction to Genetic Analysis
Chapter 16
Mechanisms of Gene Mutation
a. Which of the eight sequences would have the biggest hot spot for frameshift sequences?
b. Which of the eight sequences would have the biggest hot spot for deletions of more than a
few base pairs?
5. Suppose that an organism from outer space was analyzed and found to have DNA with six
bases, J, K, L, M, P, and Q. These bases have specific pairing partners; so, in the DNA
double helix, J pairs with K (J–K), L pairs with M (L–M), and P pairs with Q (P–Q).
a. Write out a sequence of a single strand of this DNA with more than 30 bases, and build
into it a hot spot for small mutations (of only a few base pairs or less), and explain why this
sequence would be a hotspot relative to other sequences.
b. If you had to design a 1000-base-pair gene with these six bases, what principles would
you use to prevent deletions of different segments from occurring with high frequency
throughout the gene. This includes not only short deletions but also long deletions (of
greater than 50 base pairs).
6. Describe two spontaneous lesions that can lead to mutations.See answer
7. Compare the mechanism of action of 5-bromouracil (5-BU) with ethyl methanesulfonate
(EMS) in causing mutations. Explain the specificity of mutagenesis for each agent in light
of the proposed mechanism.See answer
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An Introduction to Genetic Analysis
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Mechanisms of Gene Mutation
8. Fill in the following table, using a plus sign (+) to indicate that the mutagenic lesion (base
damage) induces the indicated base change and a minus sign (−) if it does not.
9. Compare the two different systems used to repair AP sites and to remove bulky chemical
adducts.
10. Describe the repair systems that operate after replication.See answer
11. Normal (tight) auxotrophic mutants will not grow at all in the absence of the appropriate
supplement to the medium. However, in mutant hunts for auxotrophic mutants, it is
common to find some mutants (called leaky) that grow very slowly in the absence of the
appropriate supplement but normally in the presence of the supplement. Propose an
explanation for the molecular nature of the leaky mutants.See answer
12. Strain A of Neurospora contains an ad-3 mutation that reverts spontaneously at a rate of
10−6. Strain A is crossed with a newly acquired wild-type isolate, and Ad-3 strains are
recovered from the progeny. When 28 different Ad-3 progeny strains are examined, 13
lines are found to revert at the rate of 10−6, but the remaining 15 lines revert at the rate of
10−3. Formulate a hypothesis to account for these findings, and outline an experimental
program to test your hypothesis.
13. a.
Why is it impossible to induce nonsense mutations (represented at the mRNA level
by the triplets UAG, UAA, and UGA) by treating wild-type strains with mutagens that
cause only AT → GC transitions in DNA?
b. Hydroxylamine (HA) causes only GC → AT transitions in DNA. Will HA produce
nonsense mutations in wild-type strains?
c. Will HA treatment revert nonsense mutations? See answer
14. Several auxotrophic point mutants in Neurospora are treated with various agents to see if
reversion will occur. The following results were obtained (a plus sign indicates reversion):
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An Introduction to Genetic Analysis
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Mechanisms of Gene Mutation
a. For each of the five mutants, describe the nature of the original mutation event (not the
reversion) at the molecular level. Be as specific as possible.
b. For each of the five mutants, name a possible mutagen that could have caused the
original mutation event. (Spontaneous mutation is not an acceptable answer.)
c. In the reversion experiment for mutant 5, a particularly interesting prototrophic
derivative is obtained. When this type is crossed with a standard wild-type strain, the
progeny consists of 90 percent prototrophs and 10 percent auxotrophs. Provide a full
explanation for these results, including a precise reason for the frequencies observed.
15. You are using nitrous acid to “revert” mutant nic-2 al-leles in Neurospora. You treat cells,
plate them on a medium without nicotinamide, and look for prototrophic colonies. You
obtain the following results for two mutant alleles. Explain these results at the molecular
level, and indicate how you would test your hypotheses.
a. With nic-2 allele 1, you obtain no prototrophs at all.
b. With nic-2 allele 2, you obtain three prototrophic colonies, and you cross each
separately with a wild-type strain. From the cross prototroph A × wild type, you obtain 100
progeny, all of which are prototrophic. From the cross prototroph B × wild type, you
obtain 100 progeny, of which 78 are prototrophic and 22 are nicotinamide requiring. From
the cross prototroph C × wild type, you obtain 1000 progeny, of which 996 are
prototrophic and 4 are nicotinamide requiring. See answer
16. Devise imaginative screening procedures for detecting the following mutants:
a. Nerve mutants in Drosophila.
b. Mutants lacking flagella in a haploid unicellular alga.
c. Supercolossal-sized mutants in bacteria.
d. Mutants that overproduce the black compound melanine in normally white haploid
fungus cultures.
e. Individual humans (in large populations) whose eyes polarize incoming light.
f. Negatively phototrophic Drosophila or unicellular algae.
g. UV-sensitive mutants in haploid yeast.
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Mechanisms of Gene Mutation
17. For each lesion in parts a–g, indicate which of the following repair systems repairs that
lesion.
(1) . alkyl transferase
(2). endonuclease
(3) . photolyase
(4) . MutY glycosylase
(5) . MutM glycosylase
(6) . uracil DNA glycosylase
(7) . general nucleotide excision repair
(8) . methyl-directed mismatch repair
a. Deamination of cytosine
b. 8-oxodG
c. Aflatoxin B1 adduct
d. G–T mispair as replication error
e. 5′-CC-3′ dimer
f. AP site
g. O6-methyl guanine
Chapter 16*
6. Depurination results in the loss of adenine or guanine from the nucleotide. Because the
resulting apurinic site cannot specify a complementary base, replication is blocked. Under
certain conditions, replication proceeds with a random insertion of a base opposite the
apurine site. In three-fourths of these insertions, a mutation will result.
Deamination of cytosine yields uracil. If left unrepaired, uracil will be paired with adenine
during replication, ultimately resulting in a transition mutation.
8-OxodG can pair with adenine, resulting in a transversion.
7. 5-Bromouracil is an analog of thymine. It undergoes tautomeric shifts at a higher frequency
than does thymine and, therefore, is more likely to pair with G than is thymine during
replication. At the next replication, this likelihood will lead to a GC pair rather than the
original AT pair.
Ethyl methanesulfonate is an alkylating agent that produces O-6-ethylguanine. This
alkylated guanine will mispair with thymine, which leads to an AT pair rather than a GC
pair at the next replication.
10. Mismatch repair occurs if a mismatched nucleotide is inserted during replication. The new,
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An Introduction to Genetic Analysis
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Mechanisms of Gene Mutation
incorrect base is removed, and the proper base is inserted. The enzymes required can
distinguish between new and old strands because, in E. coli, the old strand is methylated.
Recombination repair occurs if lesions such as AP sites and UV photodimers block
replication (there is a gap in the complementary strand). Recombination fills this gap with
the corresponding segment from the sister DNA molecule, which is normal in both strands.
This produces one DNA molecule with a gap across from a correct strand, which can then
be filled by complementation, and one with a photodimer across from a correct strand.
11. Leaky mutants are mutants with an altered protein product that retains a low level of
function. Enzyme activity may, for instance, be reduced rather than abolished by a
mutation.
13. a. Because 5′-UAA-3′ does not contain G or C, a transition to a GC pair in the DNA cannot
result in 5′-UAA-3′. 5′-UGA-3′ and 5′-UAG-3′ have the DNA antisense-strand sequences
of 3′-ACT-5′ and 3′-ATC-5′, respectively. A transition to either of these stop codons
occurs from the nonmutant 3′-ATT-5′. However, a DNA sequence of 3′-ATT-5′ results in
an RNA sequence of UAA, itself a stop codon.
b. Yes. An example is 5′-UGG-3′, which encodes Trp, changed to 5′-UAG-3′.
c. No. In the three stop codons, the only base that can be acted on is G (in UAG, for
instance). Replacing the G with an A would result in 5′-UAA-3′, a stop codon.
15. To understand these data, recall that half of the progeny should come from the wild-type
parent.
a. A lack of revertants suggests either a deletion or an inversion within the gene.
b. Prototroph A: Because 100 percent of the progeny are prototrophic, a reversion at the
original mutant site may have occurred.
Prototroph B: Half of the progeny are parental prototrophs, and the remaining prototrophs,
28 percent, are the result of the new mutation. Notice that 28 percent is approximately
equal to the 22 percent auxotrophs. The suggestion is that an unlinked suppressor mutation
occurred, yielding independent assortment with the nic mutant.
Prototroph C: There are 496 “revertant” prototrophs (the other 500 are parental
prototrophs) and 4 auxotrophs. This result suggests that a suppressor mutation occurred in
a site very close [100% (4 × 2)/1000 = 0.8 m.u.] to the original mutation.
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勇者并非无所畏惧，而是能判断出有比恐惧更重要的东西.