Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini Induced mutations Mutations are...

Genetica per Scienze Naturalia.a. 05-06 prof S. Presciuttini

Induced mutations MMutations are categorized as utations are categorized as inducedinduced or or spontaneous.spontaneous.IInduced nduced

mutationsmutations are defined as those that arise after purposeful treatment are defined as those that arise after purposeful treatment with with mutagensmutagens,, environmental agents that are known to increase the environmental agents that are known to increase the rate of mutations.rate of mutations.

Spontaneous mutations are those that arise in the absence of known Spontaneous mutations are those that arise in the absence of known mutagen treatment. They account for the "background rate" of mutagen treatment. They account for the "background rate" of mutation and are the ultimate source of natural genetic variation that mutation and are the ultimate source of natural genetic variation that is seen in populations.is seen in populations. The frequency at which spontaneous mutations The frequency at which spontaneous mutations occur is low, generally in the range of one cell in 10occur is low, generally in the range of one cell in 1055 to 10 to 1088..

Therefore, if a large number of mutants is required for genetic Therefore, if a large number of mutants is required for genetic analysis, mutations must be analysis, mutations must be induced.induced. The induction of mutations is The induction of mutations is accomplished by treating cells with mutagens.accomplished by treating cells with mutagens.


Spontaneous vs induced mutations Recognize that the distinction between induced and spontaneous is purely Recognize that the distinction between induced and spontaneous is purely

operational. If we are aware that an organism was operational. If we are aware that an organism was mutagenizedmutagenized,, then we infer that then we infer that any mutations that arise after this mutagenesis were induced. However, this is not any mutations that arise after this mutagenesis were induced. However, this is not true in an absolute sense. The mechanisms that give rise to spontaneous mutations true in an absolute sense. The mechanisms that give rise to spontaneous mutations also are in action in this mutagenized organism. In reality, there will always be a also are in action in this mutagenized organism. In reality, there will always be a subset of mutations recovered after mutagenesis that are independent of the action subset of mutations recovered after mutagenesis that are independent of the action of the mutagen. The proportion of mutations that fall into this subset depends on of the mutagen. The proportion of mutations that fall into this subset depends on how potent a mutagen ishow potent a mutagen is. The higher the rate of induced mutations, the lower the . The higher the rate of induced mutations, the lower the proportion of recovered mutations that are actually "spontaneous" in origin.proportion of recovered mutations that are actually "spontaneous" in origin.

Induced and spontaneous mutations Induced and spontaneous mutations arise by generally different mechanismsarise by generally different mechanisms. . After considering these mechanisms, we shall explore the subject of biological After considering these mechanisms, we shall explore the subject of biological mutation repair. Without these repair mechanisms, the rate of mutation would be so mutation repair. Without these repair mechanisms, the rate of mutation would be so high that cells would accumulate too many mutations to remain viable and capable high that cells would accumulate too many mutations to remain viable and capable of reproduction. Thus, the mutational events that do occur are those rare events that of reproduction. Thus, the mutational events that do occur are those rare events that have somehow been overlooked or bypassed by the repair processes.have somehow been overlooked or bypassed by the repair processes.


Mutation induction The task of finding rare mutations in multicellular organisms is The task of finding rare mutations in multicellular organisms is

difficult compared with that in microorganisms. difficult compared with that in microorganisms. In 1928, Hermann J. Muller devised a method of searching for any In 1928, Hermann J. Muller devised a method of searching for any

lethal mutation on the X chromosome in Drosophila.lethal mutation on the X chromosome in Drosophila. He first “constructed” an X chromosome called ClB. The C stands for a He first “constructed” an X chromosome called ClB. The C stands for a

chromosomal rearrangement called an inversion; it causes suppression of chromosomal rearrangement called an inversion; it causes suppression of crossover because, in a female fly carrying this special ClB chromosome and a crossover because, in a female fly carrying this special ClB chromosome and a normal X, the X chromosome chromatids do not recombine.normal X, the X chromosome chromatids do not recombine.

The ClB chromosome also bears l, a recessive lethal allele, and the allele B, The ClB chromosome also bears l, a recessive lethal allele, and the allele B, which determines the dominant bar-eye phenotype. C l B/Y males die because which determines the dominant bar-eye phenotype. C l B/Y males die because of hemizygosity for the lethal allele, but the chromosome can be maintained in of hemizygosity for the lethal allele, but the chromosome can be maintained in heterozygous C l B/C+l+B+ females.heterozygous C l B/C+l+B+ females.

This special This special ClBClB system allowed Muller to screen for lethal mutations system allowed Muller to screen for lethal mutations anywhere on the X chromosomes in samples of male gametes.anywhere on the X chromosomes in samples of male gametes.


Muller’s protocol

The The ClBClB test for new X chromosome mutations in test for new X chromosome mutations in Drosophila.Drosophila. The symbol The symbol mm represents a recessive lethal mutation anywhere on the X chromosome. Observing represents a recessive lethal mutation anywhere on the X chromosome. Observing presence versus absence of males in each individual progeny amounts to scanning—by presence versus absence of males in each individual progeny amounts to scanning—by genetic analysis—a sample of gametes from the male parent of the bar-eyed daughter.genetic analysis—a sample of gametes from the male parent of the bar-eyed daughter.

Bar-eyed daughters from the cross of females heterozygous for the Bar-eyed daughters from the cross of females heterozygous for the ClBClB chromosome chromosome and wild-type males are crossed individually with wild-type males. Each bar-eyed and wild-type males are crossed individually with wild-type males. Each bar-eyed daughter lays her eggs in a separate culture vial. When the progeny hatch, the vials are daughter lays her eggs in a separate culture vial. When the progeny hatch, the vials are examined for the presence of males. If there was a new lethal recessive mutation on an examined for the presence of males. If there was a new lethal recessive mutation on an X chromosome in one of the original male gametes, then the F1 female carrying that X chromosome in one of the original male gametes, then the F1 female carrying that chromosome will not produce any viable male progeny. chromosome will not produce any viable male progeny.


Increasing the mutation rates Muller found the recessive frequency of such mutations occurring Muller found the recessive frequency of such mutations occurring

spontaneously to be about 1.5 per 1000 chromosomes, still a relatively spontaneously to be about 1.5 per 1000 chromosomes, still a relatively low value for an entire chromosome.low value for an entire chromosome.

Muller then asked whether there were any agents that would increase Muller then asked whether there were any agents that would increase the rate of mutation. Using the the rate of mutation. Using the ClBClB test, he measured X-linked lethal test, he measured X-linked lethal frequencies after irradiating males with X rays and observed frequencies after irradiating males with X rays and observed frequencies that were much higher than those in unirradiated controls.frequencies that were much higher than those in unirradiated controls.

His results supplied the first experimental evidence of a mutagen—in His results supplied the first experimental evidence of a mutagen—in this case, the X rays.this case, the X rays.


Induced mutation rate It is now known that many kinds of radiation increase mutations. Radiation is often It is now known that many kinds of radiation increase mutations. Radiation is often

categorized as categorized as ionizingionizing or or nonionizing,nonionizing, depending on whether ions are produced in depending on whether ions are produced in the tissue through which it passes. X rays and the tissue through which it passes. X rays and γγ (gamma) rays, for example, do (gamma) rays, for example, do produce ions.produce ions.

The harnessing of nuclear energy has become a social issue because of the powerful The harnessing of nuclear energy has become a social issue because of the powerful mutagenic effect of nuclear radiation. No containment system is infallible, and mutagenic effect of nuclear radiation. No containment system is infallible, and recent decades have seen many examples of accidental release of nuclear isotopes. recent decades have seen many examples of accidental release of nuclear isotopes. Furthermore, disposal of nuclear wastes is not as easy as had been supposed.Furthermore, disposal of nuclear wastes is not as easy as had been supposed.

Type of radiation

Percentage of male X chromosomes bearing recessive

lethal mutations after a dose of 1000 roentgens*

Visible light (spontaneous) 0.15

X rays (25 Mev) 1.7

β rays, γ rays, hard X rays 2.9

Soft X rays 2.5

Neutrons 1.9

α rays 0.84* The roentgen (r) is a unit of radiation energy

The effects of several types of The effects of several types of radiation on increasing radiation on increasing mutation frequencies in mutation frequencies in DrosophilaDrosophila


Dose-response curvesLinear relation between X-ray dose to Linear relation between X-ray dose to which which D. melanogasterD. melanogaster were exposed and were exposed and the percentage of mutations (mainly sex-the percentage of mutations (mainly sex-linked recessive lethals) linked recessive lethals)

It is known that, within a certain It is known that, within a certain range of radiation dosage, range of radiation dosage, induction of point mutations is induction of point mutations is linear; that is, if we double or linear; that is, if we double or halve the radiation level, the halve the radiation level, the number of mutants produced will number of mutants produced will vary accordingly. vary accordingly. Radiation doses generally are Radiation doses generally are cumulative. If a population of cumulative. If a population of organisms is repeatedly exposed to organisms is repeatedly exposed to radiation, the frequency of radiation, the frequency of mutations induced will be in direct mutations induced will be in direct proportion to the total amount of proportion to the total amount of radiation absorbed over time. radiation absorbed over time.


Ionizing radiation Ionizing radiation results in the formation of ionized and excited molecules that can 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 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 of biological systems, the molecules generated by the effects of ionizing radiation on water produce the most damage. Many different types of reactive oxygen on water produce the most damage. Many different types of reactive oxygen specials are produced, including superoxide radicals, such as ·OH. The most specials are produced, including superoxide radicals, such as ·OH. The most biologically relevant reaction products are ·OH, Obiologically relevant reaction products are ·OH, O22

, and H, and H22OO22. These species can . These species can

damage bases and cause different adducts and degradation products. Among the damage bases and cause different adducts and degradation products. Among the most prevalent, which result in mutations, are thymine glycol and 8-oxodG. most prevalent, which result in mutations, are thymine glycol and 8-oxodG. Ionizing radiation can cause Ionizing radiation can cause breakage of the N-glycosydic bondbreakage of the N-glycosydic bond, leading to the , leading to the formation of AP sites, and can cause formation of AP sites, and can cause strand breaks strand breaks that are responsible for most of that are responsible for most of the lethal effects of such radiation.the lethal effects of such radiation.

DNA damage products formed after DNA damage products formed after attack by oxygen radicals. dR = attack by oxygen radicals. dR = deoxyribosedeoxyribose


Ultraviolet light Ultraviolet lightUltraviolet light generates generates ttwo different lesions that occur at wo different lesions that occur at adjacent pyrimidine adjacent pyrimidine

residuesresidues:: the cyclobutane pyrimidine photodimer and the 6-4 photoproduct. These the cyclobutane pyrimidine photodimer and the 6-4 photoproduct. These lesions interfere with normal base pairing; hence, induction of the SOS system is lesions interfere with normal base pairing; hence, induction of the SOS system is required for mutagenesis. The insertion of incorrect bases across from UV required for mutagenesis. The insertion of incorrect bases across from UV photoproducts is at the 3photoproducts is at the 3’’ position of the dimer, and more frequently for 5 position of the dimer, and more frequently for 5’’-CC-3-CC-3’’ and 5and 5’’-TC-3-TC-3’’ dimers. The C dimers. The C T transition is the most frequent mutation, but other T transition is the most frequent mutation, but other base substitutions (transversions) and frameshifts also are stimulated by UV light, base substitutions (transversions) and frameshifts also are stimulated by UV light, as are duplications and deletions. as are duplications and deletions.

(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 photoproduct. The structure forms most prevalently with 5’-CC-3’ and 5’-TC-3’, between the C-6 and the C-4 positions of two adjacent pyrimidines, causing a significant perturbation in local structure of the double helix.


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

For example, For example, 5-bromouracil5-bromouracil (5-BU) (5-BU) is an analog of thymine that has bromine at is an analog of thymine that has bromine at the C-5 position in place of the CHthe C-5 position in place of the CH33 group found in thymine. This change does not group found in thymine. This change does not

affect the atoms that take part in hydrogen bonding in base pairing, but the presence 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. of the bromine significantly alters the distribution of electrons in the base.

The normal structure (the keto form) The normal structure (the keto form) of 5-BU pairs with adenine, as of 5-BU pairs with adenine, as shown shown herehere. 5-BU can frequently . 5-BU can frequently change to either the enol form or an change to either the enol form or an ionized form; the latter pairs in vivo ionized form; the latter pairs in vivo with guanine. with guanine.


Intercalating agents The The intercalating agentsintercalating agents form another important class of DNA modifiers. These form another important class of DNA modifiers. These

agents are planar molecules, which mimic base pairs and are able to slip themselves agents are planar molecules, which mimic base pairs and are able to slip themselves in in (intercalate)(intercalate) between the stacked nitrogen bases at the core of the DNA double between the stacked nitrogen bases at the core of the DNA double helix helix (see figure)(see figure). In this intercalated position, the agent can cause single-. In this intercalated position, the agent can cause single-nucleotide-pair insertions or deletions. Intercalating agents may also stack between nucleotide-pair insertions or deletions. Intercalating agents may also stack between bases in single-stranded DNAbases in single-stranded DNA..


Bulky addition products Aflatoxin BAflatoxin B11 ((AFBAFB11)) is a powerful carcinogenis a powerful carcinogen, that may contaminate human food, that may contaminate human food. It . It

generates generates apurinicapurinic sites sites following the formation of an addition product at the N-7 following the formation of an addition product at the N-7 position of guanine. Studies with apurinic sites generated in vitro demonstrated a position of guanine. Studies with apurinic sites generated in vitro demonstrated a requirement for the SOS system and showed that the SOS bypass of these sites 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 leads to the preferential insertion of an adenine across from an apurinic site. Thus agents that cause depurination at guanine residues should preferentially induce agents that cause depurination at guanine residues should preferentially induce GCGC TA transversions. TA transversions.

AFBAFB11 is a member of a class of chemical carcinogens known as is a member of a class of chemical carcinogens known as bulky addition bulky addition

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


Specific mispairing Some mutagens are not incorporated into the DNA but instead alter a base, causing Some mutagens are not incorporated into the DNA but instead alter a base, causing

specific mispairing. Certain specific mispairing. Certain alkylating agentsalkylating agents,, such as such as ethylmethanesulfonateethylmethanesulfonate (EMS)(EMS) and the widely used and the widely used nitrosoguanidinenitrosoguanidine (NG), (NG), operate by this pathway: operate by this pathway:

Although such agents add alkyl groups (an ethyl group in EMS and a methyl group in 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 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 to the oxygen at the 6 position of guanine to create an O-6-alkylguanine. This addition leads to direct mispairing with thymine, and would result in GCaddition leads to direct mispairing with thymine, and would result in GCAT AT transitions at the next round of replication. As expected, determinations of mutagenic transitions at the next round of replication. As expected, determinations of mutagenic specificity for EMS and NG show a strong preference for GCspecificity for EMS and NG show a strong preference for GCAT transitions. AT transitions.


Forward mutation frequencies obtained with various mutagens in Neurospora

The assay measures the frequency of The assay measures the frequency of ad-3ad-3 mutants. It so happens that mutants. It so happens that such mutants are red, so they can be detected against a background of such mutants are red, so they can be detected against a background of white white adad-3-3++ colonies. colonies.

Mutagenic treatment Exposure

time (minutes) Survival (%)

Number of ad-3 mutants

per 106 survivors

No treatment (spontaneous rate) 100 ~0.4 Amino purine (15 mg/ml) During growth 100 3 Ethyl methane sulfonate (1%) 90 56 25 Nitrous acid (0.05 m) 160 23 128 X rays (2000 r/min) 18 16 259 Methyl methane sulfonate (20 mm) 300 26 350 UV rays (600 erg/mm2 per min) 6 18 375

Nitrosoguanidine (25 m) 240 65 1500 ICR-170 acridine mustard (5 g/ml) 480 28 2287


Specificity of mutagensThe distribution of mutations among 36 sites in the The distribution of mutations among 36 sites in the lacIlacI gene is shown for three gene is shown for three mutagens: mutagens: EMSEMS, , UV lightUV light, and , and aflatoxin Baflatoxin B11. The height of each bar represents the . The height of each bar represents the

number of occurrences of mutations at the respective site. Some hot spots are shown number of occurrences of mutations at the respective site. Some hot spots are shown off-scale, with the number of occurrences indicated directly above the respective peak. off-scale, with the number of occurrences indicated directly above the respective peak.

For instance, in the UV-For instance, in the UV-generated collection, one site generated collection, one site resulting from a GCresulting from a GCAT AT transition is represented by 80 transition is represented by 80 occurrences. Each mutational occurrences. Each mutational site represented in the figure site represented in the figure generates an amber (UAG) generates an amber (UAG) codon in the corresponding codon in the corresponding mRNA. The mutations are mRNA. The mutations are arranged according to the type arranged according to the type of base substitution. Asterisks of base substitution. Asterisks mark the positions of 5-mark the positions of 5-methylcytosines.methylcytosines.


Still on mutagen specificity The The previous previous graphs show the distribution of base-substitution graphs show the distribution of base-substitution

mutations that create chain-terminating UAG codons. mutations that create chain-terminating UAG codons. TThe specific he specific sequence changes are known for each sequence changes are known for each lacIlacI site, allowing the graphs to site, allowing the graphs to be broken down into each category of substitution.be broken down into each category of substitution.

The graphs The graphs reveals the two components of mutational specificity. reveals the two components of mutational specificity. First, each mutagen shown favors a specific category of substitution.First, each mutagen shown favors a specific category of substitution. For example, For example, EMS and UV favor GCEMS and UV favor GCAT transitionsAT transitions, whereas , whereas AFBAFB11 favors favors

GCGCTA transversionsTA transversions. These preferences are related to the different . These preferences are related to the different mechanisms of mutagenesis.mechanisms of mutagenesis.

Second, even within the same category, there are large differences in Second, even within the same category, there are large differences in mutation rate.mutation rate. These differences can be seen best with These differences can be seen best with UV light for the GCUV light for the GCAT changesAT changes. .

Some aspect of the surrounding DNA sequence must cause these differences. In 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 some cases, the cause of mutational hot spots can be determined by DNA sequence studies, as previously described for certain frameshift sites. In many sequence studies, as previously described for certain frameshift sites. In many examples of mutagen-induced hot spots, the precise reason for the high examples of mutagen-induced hot spots, the precise reason for the high mutability of specific sites is still unknown.mutability of specific sites is still unknown.


Mechanisms of mutagenesis Mutagens induce mutations by at least three different Mutagens induce mutations by at least three different

mechanisms.mechanisms. They can replace a base in the DNA They can replace a base in the DNA They can alter a base so that it specifically mispairs with another They can alter a base so that it specifically mispairs with another

base,base, They can damage a base so that it can no longer pair with any base They can damage a base so that it can no longer pair with any base

under normal conditions.under normal conditions.

Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini Induced mutations Mutations are...

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