DNA Mutation and Repair

DNA
Primary function permanent storage of information Does not normally change Mutations do occur

 Mutation
Heritable change in the genetic material Permanent structural change of DNA
Alteration can be passed on to daughter cells (somatic cell) Mutations in reproductive cells can be passed to offspring (germ line)

SOMATIC VS. GERM-LINE

The timing of mutations in multicellular organisms plays an important role
Mutations may occur in gametes or a fertilized egg Mutations may occur later in life
Embryonic or adult stages

Timing can affect

The severity of the genetic effect The ability to be passed from parent to offspring
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SOMATIC VS. GERM-LINE

Animals possess germ-line and somatic cells
Germ-line cells
Cells giving rise to gametes

Somatic cells
All cells of the body excluding the germ-line cells
e.g., Muscle cells, nerve cells, etc.

SOMATIC VS. GERM-LINE

Germ-line cells
Germ-line mutations can occur in gametes Germ-line mutations can occur in a precursor cell that produces gametes All cells in the resulting offspring will contain the mutation
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SOMATIC VS. GERM-LINE

Somatic cells
Somatic mutations in embryonic cells can result in patches of tissues containing the mutation
Size of the patch depends on the timing of the mutation Individual is a genetic mosaic

 Mutations
Provide allelic variation
Ultimate source of genetic variation Foundation for evolutionary change

Various phenotypic effects

Neutral Harmful Beneficial

 Mutations
Many inherited diseases result from mutated genes Diseases such as various cancers can be caused by environmental agents known to cause DNA mutations
Mutagens

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MODEL ORGANISMS
Much of our understanding of mutations is a result of the study of model organisms
e.g., Bacteria, yeast, Drosophila, etc.
Amenable to analysis Short generation time, numerous offspring, etc.

Often exposed to mutagenic environmental agents

Effects of mutations are studied

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TYPES OF MUTATIONS
Types of mutations
Chromosome mutations
Changes in chromosome structure

Genome mutations
Changes in chromosome number

Single-gene mutations
Relatively small changes in DNA structure Occur within a particular gene Focus of study in this chapter
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TYPES OF MUTATIONS
Mutations involve the permanent alteration of a DNA sequence
Alteration of base sequence Removal or addition of one or more nucleotides

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MUTATIONS
Point mutations
Change in a single base pair within the DNA Two main types of point mutations
Base substitutions
Transition Transversion

Small deletions or insertions

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MUTATIONS
Two types of base substitutions
Transition
Pyrimidine changed to another pyrimidine
e.g., C T

Purine changed to another purine

e.g., A G

Transversion
Purines and pyrimidines are interchanged
e.g., A C

More rare than transitions

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EFFECTS OF MUTATIONS
What can happen when a mutation occurs in the DNA (e.g. sickle cell anemia)

Figure 1. Concept of a mutation in the protein-coding region of a gene. (Note that not all mutations lead to altered proteins and that not all mutations are in protein-coding regions.)

EFFECTS OF MUTATIONS
Mutations within the coding sequence of a gene can have various effects on the encoded polypeptides amino acid sequence
Silent mutations Missense mutations
Included neutral mutations

Nonsense mutations Frameshift mutations

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 Silent mutations
Amino acid sequence is not altered
e.g., CCC CCG (pro pro)
Genetic code is degenerate Alterations of the third base of a codon often do not alter the encoded amino acid

Phenotype is not affected

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EFFECTS OF MUTATIONS
Missense mutations
Amino acid sequence is altered
e.g., GAA GTA (glu val)

Phenotype may be affected

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EFFECTS OF MUTATIONS
Neutral mutations
Type of missense mutation Amino acid sequence is altered
e.g., CTT ATT (leu ile) e.g., GAA GAC (glu asp)

No detectable effect on protein function

Missense mutations substituting an amino acid with a similar chemistry to the original is likely to be neutral
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EFFECTS OF MUTATIONS
Nonsense mutations
Normal codon is changed into a stop codon
e.g., AAA AAG (lys stop)

Translation is prematurely terminated

Truncated polypeptide is formed

Protein function is generally affected

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A nonsense mutation and its effect on translation

EFFECTS OF MUTATIONS

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EFFECTS OF MUTATIONS

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EFFECTS OF MUTATIONS
Mutations occasionally produce a polypeptide with an enhanced ability to function
Relatively rare May result in an organism with a greater likelihood to survive and reproduce Natural selection may increase the frequency of this mutation in the population
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MUTATION TYPES
Genetic terms to describe mutations
Wild-type
Relatively common genotype Generally the most common allele

Variant
Mutant allele altering an organisms phenotype

Forward mutation
Changes wild-type allele into something else

Reverse mutation
Reversion Restores wild-type allele
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MUTATION TYPES
Genetic terms to describe mutations
Deleterious mutation
Decreases an organisms chance of survival

Lethal mutation
Results in the death of an organism Extreme example of a deleterious mutation

Conditional mutants
Affect the phenotype only under a defined set of conditions e.g., Temperature-sensitive (ts) mutants

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MUTATION TYPES
Genetic terms to describe mutations
Suppressor mutation
Second mutation that restores the wild-type phenotype Intragenic suppressor
Secondary mutation in the same gene as the first mutation Differs from a reversion Second mutation is at a different site than the first

Intergenic suppressor
Secondary mutation in a different gene than the first mutation
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MUTATION TYPES
Two general types of intergenic suppressors
Those involving an ability to defy the genetic code Those involving a mutant structural gene

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MUTATION TYPES
Intergenic suppressor mutations involving an ability to defy the genetic code
e.g., tRNA mutations
Altered anticodon region e.g., Recognize a stop codon
May suppress a nonsense mutation in a gene May also suppress stop codons in normal genes

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MUTATION TYPES
Intergenic suppressors involving a mutant structural gene
Usually involve altered expression of one gene that compensates for a loss-of-function mutation affecting another gene
Second gene may take over the functional role of the first May involve proteins participating in a common cellular function

Sometimes involve mutations in genetic regulatory proteins

e.g., Transcription factors activating other genes that can compensate for the mutation in the first gene

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A tRNA Tyr gene has mutated so that the tRNAs anticodon is changed from 3-AUG-5 to 3-AUC-5, which can read a UAG nonsense codon, inserting tyrosine in the polypeptide chain at that codon.

TRINUCLEOTIDE REPEATS
DNA trinucleotide repeats
Three nucleotide sequences repeated in tandem
e.g., CAGCAGCAGCAGCAGCAG Generally transmitted normally from parent to offspring without mutation

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TRINUCLEOTIDE REPEATS
Trinucleotide repeat expansion (TNRE)
Number of repeats can readily increase from one generation to the next Cause of several human genetic diseases
Length of a repeat has increased above a certain critical size Becomes prone to frequent expansion

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TRINUCLEOTIDE REPEATS
TNRE disorders
Fragile X syndrome (FRAXA) FRAXE mental retardation Myotonic muscular dystrophy (DM) Spinal and bulbar muscular atrophy (SBMA) Huntington disease (HD) Spinocerebellar ataxia (SCA1)

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TRINUCLEOTIDE REPEATS
TNRE disorders

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TRINUCLEOTIDE REPEATS
TNRE disorders
Expansion may be within a coding sequence of a gene
Most expansions are of a CAG repeat Encoded proteins possess long tracts of glutamine
CAG encodes a glutamine codon

Presence of glutamine tracts causes aggregation of the proteins Aggregation is correlated with the progression of the disease
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TRINUCLEOTIDE REPEATS
TNRE disorders
Expansion may be in a noncoding region of a gene
Two fragile X syndromes
Repeat produces CpG islands that become methylated Methylation can lead to chromosome compaction Can silence gene transcription

Myotonic muscular dystrophy

Expansions may cause abnormal changes in RNA structure

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TRINUCLEOTIDE REPEATS
TNRE disorders
Severity of the disease tends to worsen in future generations
Anticipation

Severity of the disease depends on the parent from whom it was inherited
e.g., In Huntingdon disease, TNRE likely to occur if mutation gene is inherited from the father e.g., In myotonic muscular dystrophy, TNRE likely to occur if mutation gene is inherited from the mother
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TRINUCLEOTIDE REPEATS
TNRE disorders

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TRINUCLEOTIDE REPEATS
TNRE disorders
Cause of TNRE is not well understood Trinucleotide repeat may produce alterations in DNA structure
e.g., Stem-loop formation May lead to errors in DNA replication

TNRE within certain genes alters gene expression

Disease symptoms are produced

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CHROMOSOME STRUCTURE
Altered chromosome structure can alter gene expression
Inversions and translocations commonly have no obvious phenotypic effects Phenotypic effects sometimes occur
Position effect

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CHROMOSOME STRUCTURE
Altered chromosome structure can alter gene expression and phenotype
Breakpoint may occur within a gene
Expression of the gene is altered

Breakpoint may occur near a gene

Expression is altered when moved to a new location May be moved next to regulatory elements influencing the expression of the relocated gene
i.e., Silencers or enhancers

May reposition a gene from a euchromatic region to a highly condensed (heterochromatic) region
Expression may be turned off
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CAUSES OF MUTATIONS
Two causes of mutations
Spontaneous mutations
Result from abnormalities in biological processes Underlying cause lies within the cell

Induced mutations
Caused by environmental agents Cause originates outside of the cell

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Spontaneous Chemical Changes

Figure 7.9 (a) Deamination of cytosine to uracil. (b) Deamination of 5-methylcytosine (5mC) to thymine.

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CAUSES OF MUTATIONS
Induced mutations are caused by mutagens
Chemical substances or physical agents originating outside of the cell Enter the cell and then alter the DNA structure

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CAUSES OF MUTATIONS
Spontaneous mutations are random events
Not purposeful Mutations occur as a matter of chance
Some individuals possess beneficial mutations
Better adapted to their environment Increased chance of surviving and reproducing

Natural selection results in differential reproductive success

The frequency of such alleles increases in the population

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 Spontaneous mutations: Depurination

Most common type of naturally occurring chemical change Reaction with water removes a purine (A or G) from the DNA
Apurinic site

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 Spontaneous mutations: Depurination

~10,000 purines lost per 20 hours at 37oC in a typical mammalian cell
Rate of loss increased by agents causing certain base modification
e.g., Attachment of alkyl (methyl, ethyl, etc.) groups

Generally recognized by DNA repair enzymes

Mutation may result if repair system fails
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 Spontaneous mutations: Deamination of cytosines

Methylation of cytosine occurs in many eukaryotic species as well as prokaryotes Removal of an amino group from the 5-methyl cytosine produces thymine DNA repair enzymes cannot determine which is the incorrect base
Hot spots for mutations are produced

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 Spontaneous mutations: Tautomeric shifts

Common, stable form of T and G is the keto form
Interconvert to an enol form at a low rate

Common, stable form of A and C is the amino form

Interconvert to an imino form at a low rate

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 Spontaneous mutations: Tautomeric shifts

Enol and imino forms do not conform to normal base-pairing rules
AC and GT base pairs are formed

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 Certain non-mutagenic chemicals can be altered to a mutagenically active form after ingestion
Cellular enzymes such as oxidases can activate some mutagens

Certain foods contain chemicals acting as antioxidants

Antioxidants may be able to counteract the effects of mutagens and lower cancer rates
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CAUSES OF MUTATIONS
Mutagens alter DNA structure in various ways
Nitrous acid (HNO3) replaces amino groups with keto groups
-NH2 =O Can change cytosine to uracil
Pairs with A, not G

Can change adenine to hypoxanthine

Pairs with C, not T
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 Mutagens alter DNA structure in various ways

Alkylating agents covalently attach methyl or ethyl groups to bases
e.g., Nitrogen mustards, ethyl methanesulfonate (EMS)

Appropriate base pairing is disrupted

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CAUSES OF MUTATIONS
Mutagens alter DNA structure in various ways
Some mutagens directly interfere with the DNA replication process e.g., Acridine dyes such as proflavin
Flat, planar structures interchelate into the double helix
Sandwich between adjacent base pairs

Helical structure is distorted Single-nucleotide additions and deletions can result

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CAUSES OF MUTATIONS
Mutagens alter DNA structure in various ways
Some mutagens are base analogs
e.g., 2-aminopurine e.g., 5-bromouracil (5BU) Become incorporated into daughter strands during DNA replication

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CAUSES OF MUTATIONS
Mutagens alter DNA structure in various ways
Some mutagens are base analogs
5-bromouracil (5BU) is a thymine analog
Incorporated in place of thymine

5BU can base-pair with adenine

Can tautomerize and base-pair with guanine at a relatively high rate

AT A5BU G5BU GC
Transition mutations occur
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CAUSES OF MUTATIONS
Mutagens alter DNA structure in various ways
DNA molecules are sensitive to physical agents such as radiation
e.g., Ionizing radiation such as X rays and gamma rays
Short wavelength and high energy Can penetrate deeply into biological materials Creates free radicals Chemically reactive molecules Free radicals alter DNA structure in a variety of ways Deletions, single nicks, cross-linking, chromosomal breaks

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CAUSES OF MUTATIONS
Mutagens alter DNA structure in various ways
DNA molecules are sensitive to physical agents such as radiation
e.g., Nonionizing radiation such as UV light
Contains less energy Penetrates only the surface of material such as the skin Causes the formation of thymine dimers May be repaired through one of numerous repair systems May cause a mutation when that DNA strand is replicated
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Effect of UV light on base DNA

CAUSES OF MUTATIONS
Many different kinds of testes can determine if an agent is mutagenic
Ames test is commonly used
Developed by Bruce Ames

Uses his- strains of Salmonella typhimurium

Mutation is due to a point mutation rendering an enzyme inactive

Reversions can restore his+ phenotype

Ames test monitors rate of reversion mutations
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CAUSES OF MUTATIONS
Ames test
Suspected mutagen is mixed with rat liver extract and his- Salmonella typhimurium
Rat liver extract provides cellular enzymes that may be required to activate a mutagen

Bacteria are plated on minimal media

his+ revertants can be detected Mutation frequency calculated
Compared to control
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DNA REPAIR
Most mutations are deleterious
DNA repair systems are vital to the survival Bacteria possess several different DNA repair systems
Absence of a single system greatly increases mutation rate
Mutator strains

Humans defective in a single DNA repair system may manifest various disease symptoms
e.g., Higher risk of skin cancer
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DNA REPAIR
Living cells contain several DNA repair systems
Able to fix different types of DNA alterations

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