Chapter 12Molecular

Mechanisms of Mutation and DNA

Repair

Mutations• A mutation is any heritable change in the genetic

material

• Mutations are classified in a variety of ways

• Most mutations are spontaneous—they are random, unpredictable events

• Each gene has a characteristic rate of spontaneous mutation, measured as the probability of a change in DNA sequence in the time span of a single generation

Table 12.01: Major types of mutations and their distinguishing features.

• Rates of mutation can be increased by treatment with a chemical mutagen or radiation, in which case the mutations are said to be induced

• Mutations in cells that form gametes are germ-line mutations; all others are somatic mutations

• Germ-line mutations are inherited; somatic mutations are not

• A somatic mutation yields an organism that is genotypically a mixture (mosaic) of normal and mutant tissue

Mutations

• Among the mutations that are most useful for genetic analysis are those whose effects can be turned on or off by the researcher

• These are conditional mutations: they produce phenotypic changes under specific (permissive conditions) conditions but not others (restrictive conditions)

• Temperature-sensitive mutations: conditional mutation whose expression depends on temperature

Mutations

Figure 12.1: Siamese cat

Courtesy of Jen Vertullo

• Mutations can also be classified according to their effects on gene function:

A loss-of-function mutation (a knockout or null) results in complete gene inactivation or in a completely nonfunctional gene product

A hypomorphic mutation reduces the level of expression of a gene or activity of a product.

Mutations

Mutations

A hypermorphic mutation produces a greater-than-normal level of gene expression

A gain-of-function mutation qualitatively alters the action of a gene. For example, a gain-of-function mutation may cause a gene to become active in a type of cell or tissue in which the gene is not normally active.

Mutations can also be classified according to their effects on gene function:

Figure 02B: A wing with eye tissue growing out from it

Reproduced from G. Halder, P. Callaerts, and W. J. Gehring, Science 267 (1995): 1788-1792. Reprinted with permission from AAAS. [http://www.sciencemag.org/].

Figure 02D: Middle leg with an eye outgrowth at the base of the tibia

Reproduced from G. Halder, P. Callaerts, and W. J. Gehring, Science 267 (1995): 1788-1792. Reprinted with permission from AAAS. [http://www.sciencemag.org/].

• Mutations result from changes in DNA

• A base substitution replaces one nucleotide pair with another

• Transition mutations replace one pyrimidine base with the other or one purine base with the other. There are four possible transition mutations

Mutations

• Transversion mutations replace a pyrimidine with a purine or the other way around. There are eight possible transversion mutations

• Spontaneous base substitutions are biased in favor of transitions

• Among spontaneous base substitutions, the ratio of transitions to transversions is approximately 2:1

Mutations

• Mutations in protein-coding regions can change an amino acid, truncate the protein, or shift the reading frame:

• Missense or nonsynonymous substitutions result in one amino acid being replaced with another

• Synonymous or silent substitutions in DNA do not change the amino acid sequence

• Silent mutations are possible because the genetic code is redundant

Mutations

• A nonsense mutation creates a new stop codon

• Frameshift mutations shift the reading frame of the codons in the mRNA

• Any addition or deletion that is not a multiple of

three nucleotides will produce a frameshift

Mutations

Sickle-cell anemia• The molecular basis of sickle-cell anemia is a mutant

gene for b-globin

• The sickle-cell mutation changes the sixth codon in the coding sequence from the normal GAG, which codes for glutamic acid, into the codon GUG, which codes for valine

• Sickle-cell anemia is a severe genetic disease that often results in premature death

• The disease is very common in regions where malaria is widespread because it confers resistance to malaria

Figure 12.2: Molecular basis of sickle-cell anemia

Trinucleotide repeats• Genetic studies of an X-linked form of mental

retardation revealed a class of mutations called dynamic mutations because of the extraordinary genetic instability of the region of DNA involved

• The molecular basis of genetic instability is a trinucleotide repeat expansion due to the process called replication slippage

Figure 12.05: Model of replication slippage.

Fragile-X Syndrome• The X-linked condition, is associated with a class of X

chromosomes that tends to fracture in cultured cells that are starved for DNA precursors

• They are called fragile-X chromosomes, and the associated form of mental retardation is the fragile-X syndrome

• The fragile-X syndrome affects about 1 in 2500 children• The molecular basis of the fragile-X chromosome has

been traced to the expansion of a CGG trinucleotide repeat present at the site where the breakage takes place

Figure 12.3: Pedigree showing transmission of the fragile-X syndrome

Adapted from C. D. Laird, Genetics 117 (1987): 587-599.

Fragile-X Syndrome

• Normal X chromosomes have 6–54 tandem copies of CGG, whereas affected persons have 230–2300 or more copies

• An excessive number of copies of the CGG repeat cause loss of function of a gene designated FMR1(fragile-site mental retardation-1)

• Most fragile-X patients exhibit no FMR1 mRNA

• The FMR1 gene is expressed primarily in the brain and testes

Figure 12.04: Dynamic mutation in the CGG repeat.

Dynamic Mutations and Diseases

• Other genetic diseases associated with dynamic mutation include: The neurological disorders myotonic dystrophy

(with an unstable repeat of CTG) Kennedy disease (AGC) Friedreich ataxia (AAG) Spinocerebellar ataxia type 1 (AGC) Huntington disease (AGC)

Transposable Elements• In a 1940s study of the genetics of kernel mottling in

maize, Barbara McClintock discovered a genetic element that could move (transpose) within the genome and also caused modification in the expression of genes at or near its insertion site.

• Since then, many transposable elements (TEs) have been discovered in prokaryotes and eukaryotes

• They are grouped into “families” based on similarity in DNA sequence

Figure 12.F06: Sectors of purple and yellow tissue in the endosperm of maize kernels resulting from the presence of the transposable elements Ds and Ac.

Courtesy of Jerry L. Kermicle, Professor Emeritus, University of Wisconsin at Madison.

Transposable Elements

• The genomes of most organisms contain multiple copies of each of several distinct families of TEs

• Once situated in the genome, TEs can persist for long periods and undergo multiple mutational changes

• Approximately 50% of the human genome consists of TEs; most of them are evolutionary remnants no longer able to transpose

Transposable Elements• Some transposable elements transpose via a DNA

intermediate others via an RNA intermediate

• A target-site duplication is characteristic of most TEs insertions, and it results from asymmetrical cleavage of the target sequence

• A large class of TEs called DNA transposons transpose via a cut-and-paste mechanism: the TE is cleaved from one position in the genome and the same molecule is inserted somewhere else

Figure 12.07: The sequence arrangement of a cut-and-paste transposable element and the changes that take place when it inserts into the genome.

Transposable Elements• Each family of TEs has its own transposase—an

enzyme that determines distance between the cuts made in the target DNA strands

• Characteristic of DNA TEs is the presence of short terminal inverted repeats

• Another large class of TEs possess terminal direct repeats, 200–500 bp in length, called long terminal repeats, or LTRs

Transposable Elements• TEs with long terminal repeats are called LTR

retrotransposons because they transpose using an RNA transcript as an intermediate

• Among the encoded proteins is an enzyme known as reverse transcriptase, which can “reverse-transcribe,” using the RNA transcript as a template for making a complementary DNA daughter strand

• Some retrotransposable elements have no terminal repeats and are called non-LTR retrotransposons

Figure 12.09: Sequence organization of a copia transposable element in Drosophila melanogaster.

Transposable Elements

• TEs can cause mutations by insertion or by recombination

• In Drosophila, about half of all spontaneous mutations that have visible phenotypic effects result from insertions of TEs

• Genetic aberrations can also be caused by recombination between different (nonallelic) copies of a TE

Figure 12.10: Recombination between transposable elements

Figure 12.11: Unequal crossing-over

Spontaneous Mutations

• Mutations are statistically random events—there is no way of predicting when, or in which cell, a mutation will take place

• The mutational process is also random in the sense that whether a particular mutation happens is unrelated to any adaptive advantage it may confer on the organism in its environment

• A potentially favorable mutation does not arise because the organism has a need for it

Spontaneous Mutations• Several types of experiments showed that adaptive

mutations take place spontaneously and were present at low frequency in the population even before it was exposed to the selective agent

• One experiment utilized a technique developed by Joshua and Esther Lederberg called replica plating

• Selective techniques merely select mutants that preexist in a population

Figure 12.12: Replica plating.

Figure1 2.13: The ClB method for estimating the rate at which spontaneous recessive lethal mutations arise

Mutation Hot Spots

• Mutations are nonrandom with respect to position in a gene or genome

• Certain DNA sequences are called mutational hotspots because they are more likely to undergo mutation than others

• For instance, sites of cytosine methylation are usually highly mutable

Figure 12.14: Spontaneous loss of the amino group

Mutagenes• Almost any kind of mutation that can be induced by a

mutagen can also occur spontaneously, but mutagens bias the types of mutations that occur according to the type of damage to the DNA that they produce

Table 12.03: Major agents of mutation and their mechanisms of action.

Figure 12.15: Depurination

Figure 12.16: Deamination of adenine results in hypoxanthine

Figure 12.17: Mispairing mutagenesis by 5-bromouracil

Figure 12.18: Two pathways for mutagenesis by 5-bromouracil (Bu)

Figure 12.19: Chemical structures of two highly mutagenic alkylating agents

Figure 12.20: Mutagenesis of guanine by ethyl methanesulfonate (EMS)

Figure 12.21: Structural view of the formation of a thymine dimer

Figure 12.22: The relationship between the percentage of X-linked recessive lethals in D. melanogaster and x-ray dose.

DNA Repair Mechanisms

• Many types of DNA damage can be repaired• Mismatch repair fixes incorrectly matched base pairs• The AP endonuclease system repairs nucleotide sites

at which the base has been lost• Special enzymes repair damage caused to DNA by

ultraviolet light• Excision repair works on a wide variety of damaged

DNA• Postreplication repair skips over damaged bases

Mismatch Repair• Mismatch repair fixes incorrectly matched base pairs:

a segment of DNA that contains a base mismatch excised and repair synthesis followed

• The mismatch-repair system recognizes the degree of methylation of a strand and preferentially excises nucleotides from the undermethylated strand

• This helps ensure that incorrect nucleotides incorporated into the daughter strand in replication will be removed and repaired.

Table 12.6: Types of DNA damage and mechanism of repair

Mismatch Repair

• The most important role of mismatch repair is as a “last chance” error-correcting mechanism in replication

Figure 12.24: Summary of rates of error in DNA polymerization,

proofreading, and postreplication mismatch repair.

Mismatch Repair

• The daughter strand is always the undermethylated strand because its methylation lags somewhat behind the moving replication fork

Figure 12.25: Mismatch repair.

AP Repair• Deamination of cytosine creates uracil, which is

removed by DNA uracil glycosylase from deoxyribose sugar. The result is a site in the DNA that lacks a pyrimidine base (an apyrimidinic site)

• Purines in DNA are somewhat prone to hydrolysis, which leave a site that is lacking a purine base (an apurinic site)

• Both apyrimidinic and apurinic sites are repaired by a system that depends on an enzyme called AP endonuclease

Figure 12.26: Base-excision.

Figure 12.27: Action of AP endonuclease

Excision Repair• Excision repair is a

ubiquitous, multistep enzymatic process by which a stretch of a damaged DNA strand is removed from a duplex molecule and replaced by resynthesis using the undamaged strand as a template

Figure 12.28: Mechanism of nucleotide excision repair of damage to DNA.

Postreplication repair• Sometimes DNA damage

persists rather than being reversed or removed, but its harmful effects may be minimized. This often requires replication across damaged areas, so the process is called postreplication repair

Figure 12.29: Postreplication repair.

Ames Test• In view of the increased number of chemicals used

and present as environmental contaminants, tests for the mutagenicity of these substances has become important

• Furthermore, most agents that cause cancer (carcinogens) are also mutagens, and so mutagenicity provides an initial screening for potential hazardous agents

• A genetic test for mutations in bacteria that is widely used for the detection of chemical mutagens is the Ames test

Ames test• In the Ames test for mutation, histidine-requiring

(His-) mutants of the bacterium Salmonella typhimurium, containing either a base substitution or a frameshift mutation, are tested for backmutation reversion to His+

• In addition, the bacterial strains have been made more sensitive to mutagenesis by the incorporation of several mutant alleles that inactivate the excision-repair system and that make the cells more permeable to foreign molecules