gene mutation

GENE MUTATIONAND DNA REPAIR

ByM.Vharshini

Sri Ramachandra University 1

GENETIC MATERIAL• DNA

– Primary function permanent storage of information

– Does not normally change– Mutations do occur

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MUTATIONS• Mutation

– Heritable change in the genetic material

– Permanent structural change of DNA

• Alteration can be passed on to daughter cells

• Mutations in reproductive cells can be passed to offspring

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MUTATIONS• Mutations

– Provide allelic variation• Ultimate source of genetic variation• Foundation for evolutionary change

– Various phenotypic effects• Neutral• Harmful• Beneficial

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– Most mutations are neutral– More likely to be harmful than beneficial

to the individual• More likely to disrupt function than improve

function

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– 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 transitions11

EFFECTS OF MUTATIONS• Mutations within the coding sequence

of a gene can have various effects on the encoded polypeptide’s amino acid sequence– Silent mutations– Missense mutations

• Included neutral mutations– Nonsense mutations– Frameshift mutations

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EFFECTS OF MUTATIONS• 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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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 organism’s phenotype

– Forward mutation• Changes wild-type allele into something else

– Reverse mutation• “Reversion”• Restores wild-type allele

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– Deleterious mutation• Decreases an organism’s 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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– 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

mutation22

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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MUTATION TYPES• Mutations occurring outside of coding sequences

can influence gene expression– Mutations may alter the core promoter sequence

• Up promoter mutations– Mutant promoter becomes more like the

consensus sequence– Rate of transcription may be increased

• Down promoter mutations– Mutant promoter becomes less like the

consensus sequence– Affinity for regulatory factors is decreased– Rate of transcription may be decreased

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MUTATION TYPES• Mutations occurring outside of coding

sequences can influence gene expression– Mutations may alter other regulatory

sequences• lacOC mutations prevent binding of

the lac repressor– Lac operon is constituently expressed,

even in the absence of lactose» Such expression is wasteful» Such mutants are at a selective

disadvantage

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sequences can influence gene expression– Mutations may alter splice junctions

• Altered order and/or number of exons in the mRNA

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sequences can influence gene expression– Mutations may affect an

untranslated region of mRNA• 5’- or 3’-UTR• May affect mRNA stability• May affect the ability of the

mRNA to be translated

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MUTATION TYPES

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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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– 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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– 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

structure36


– 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 37


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– 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 41

CHROMOSOME STRUCTURE• Altered chromosome

structure can alter gene expression and phenotype– An eye color gene relocated to

a heterochromatic region can display altered expression

• Gene is sometimes inactivated• Variegated phenotype results

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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.

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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

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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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CAUSES OF MUTATIONS• Causes of spontaneous mutations

– Abnormalities in crossing over– Aberrant segregation of chromosomes during

meiosis– Mistakes by DNA polymerase during

replication– Alteration of DNA by chemical products of

normal metabolic processes– Integration of transposable elements– Spontaneous changes in nucleotide structure48

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•

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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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CAUSES OF MUTATIONS• Joshua and Ester Lederberg (1950s)

– Interested in the relationship between mutation and the environmental conditions shat select for mutations

• Scientists were unsure of the relationship• Two competing hypotheses

– Directed mutation hypothesis» Some scientists still believed that selective conditions

could promote specific mutations– Random mutation theory

» Mutations occur at random» Environmental factors affecting survival select for

those possessing beneficial mutations52

CAUSES OF MUTATIONS• Mutation rate

– Likelihood that a gene will be altered by a new mutation

– Expressed as the number of new mutations in a given gene per generation

• Generally 1/100,000 – 1/billion– 10-5 – 10-9

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– Mutation rate is not a constant number• Can be increased by environmental

mutagens– Induced mutations can increase beyond

frequency of spontaneous mutations

• Mutation rates vary extensively between species

– Even vary between strains of the same species

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– Some genes mutate at a much higher rate than other genes

• Some genes are longer than others• Some locations are more susceptible to

mutation– Even single genes possess mutation

hot spots» More likely to mutate than other

regions

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CAUSES OF MUTATIONS• Mutation frequency

– Number of mutant alleles of a given gene divided by the number of alleles within a population

– Timing of mutations influences mutation frequency

• Timing does not influence mutation rate– Mutation frequency depends both on mutation

rate and timing of mutations– Natural selection and genetic drift can further

increase mutation frequencies 56

CAUSES OF MUTATIONS• 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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CAUSES OF MUTATIONS• 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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CAUSES OF MUTATIONS• Spontaneous mutations: Deamination of

cytosines– Other bases are not readily deaminated– Removal of an amino group from the cytosine

base• Uracil is produced

– DNA repair enzymes generally remove this base• Uracil is recognized as an inappropriate base

– Mutation may result if repair system fails• Uracil hydrogen bonds with A, not G

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CAUSES OF MUTATIONS• 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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CAUSES OF MUTATIONS• 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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CAUSES OF MUTATIONS• 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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CAUSES OF MUTATIONS• Spontaneous mutations: Tautomeric

shifts– Tautomeric shifts immediately prior to

DNA replication can cause mutations• Resulting mismatch could be repaired• Mutation may result if repair system fails

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CAUSES OF MUTATIONS• Hermann Muller (1927)

– Showed that X rays can cause induced mutations

• Reasoned that a mutagenic agent might form defective alleles

• Experimental approach focused on formation and detection of X-linked genes in Drosophila melanogaster

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CAUSES OF MUTATIONS• The public is concerned about

mutagens for two important reasons– Mutagenic agents are often involved in

the development of human cancers– Avoiding mutations that may have

harmful effects on future offspring is desirable

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CAUSES OF MUTATIONS• An enormous array of agents can act

as mutagens– Chemical agents and physical agents

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CAUSES OF MUTATIONS• 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 rates67

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 T68

CAUSES OF MUTATIONS• 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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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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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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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 74

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 cancer77

DNA REPAIR• Living cells contain several DNA repair

systems– Able to fix different types of DNA alterations

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gene mutation

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