mutation 4th

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MUTATION


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Introduction

Mutations can happen at any time and in any cell

The phenotypic effects can range from minoralterations that are detectable only bybiochemical methods to drastic changes in

essential processes that cause, at one extreme,unrestrained cell proliferation (cancer) or, at theother extreme, the death of the cell or organism

The effect of a mutation is determined by the typeof cell containing the mutant allele, by the stage inthe life cycle ordevelopment of the organism thatthe mutation affects, and, in diploid organisms, bythe dominance or recessiveness of the mutant

allele


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Definition

A mutation is a change in a DNA base-pair(substitution, deletion, or insertion of a basepair) or a chromosome (deletion, insertion,

or rearrangement)a.Somatic mutations affect only the individual in

which they arise

b.Germ-line mutations alter gametes (tissues that

produces eggs & sperm) and passed the next

generation


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What can happen when a mutation occurs in the DNA

(e.g. sickle cell anemia)

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)


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Mutations are quantified in two different ways:

a. Mutation rate is the probability of a particular

kind of mutation as a function of time (e.g.,

number per gene per generation) or

# mutation per nucleotide pair or gene pergeneration

b. Mutation frequency is number of times a

particular mutation occurs in proportion to the

number of cells or individuals in a population(e.g., number per 100,000 organisms) or

# of a particular mutation per 100,000

organisms


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Types of mutations

Two types of point mutations:1. Base pair substitutions

a. Transitions

Convert a purine-pyrimidine to the other purine-

pyrimidine (e.g., AT to GC or TA to CG)

4 types of transitions; A G and T C

Most transitions results in synonymous substitution

b. Transversions

Convert a purine-pyrimidine to a pyrimidine-purine (e.g.,

AT to TA, or AT to CG)

8 types of transversions; A T, G C, A C, and G

T

Transversion more likely to result in nonsyn substitution


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2. Base pair deletions and insertions

can change the reading frame of the mRNA

downstream of the mutation, resulting in a

frameshift mutation.

a. When the reading frame is shifted, incorrect amino

acids are usually incorporated.

b. Frameshifts may bring stop codons into the reading

frame, creating a shortened protein.

c. Frameshifts may also result in read-through of stop

codons, resulting in a longer protein.

d. Frameshift mutations result from insertions ordeletions when the number of affected base pairs is

not divisible by three.


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Types of base pair substitutions and mutations


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Types of base pair substitutions and mutations


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Effect of a nonsense mutation on translation


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Types of mutations in ORFs:

Nonsynonymous/missense mutation

Base pair substitution results in substitution of a different amino

acid.

Nonsense mutation

Base pair substitution results in a stop codon (and shorter

polypeptide).

Neutral nonsynonymous mutationBase pair substitution results in substitution of an amino acid

with similar chemical properties (protein function is not altered).

Synonymous/silent mutation

Base pair substitution results in the same amino acid.

Frameshift mutations:

Deletions or insertions (not divisible by 3) result in translation of

incorrect amino acids, stops codons (shorter polypeptides),or

read-through of stop codons (longer polypeptides).


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The nine codons that can result from a single base change in the tyrosine codon UAU. Blue

arrows indicate transversions, gray arrows, transitions. Tyrosine codons are in boxes. Two

possible stop (''nonsense") codons are shown in red. Altogether, the codon UAU allows for six

possible missense mutations, two possible nonsense mutations, and one silent mutation


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Reverse Mutations and Suppressor

Mutations

1. Point mutations are divided into two classes based on their effect on

phenotype:

a. Forward mutations change the genotype from wild type to mutant.

b. Reverse mutations (reversions or back mutations) change the genotype from mutant

to wild-type or partially wild-type.

i. A reversion to the wild-type amino acid in the affected protein is a true reversion.ii. A reversion to some other amino acid that fully or partly restores protein function

is a partial reversion.

2. Suppressor mutations occur at sites different from the original mutation, and

mask or compensate for the initial mutation without actually reversing it.

Suppressor mutations have different mechanisms depending on the site at

which they occur.

a. Intragenic suppressors occur within the same gene as the original mutation, but at a

different site. Two different types occur:

i. A different nucleotide is altered in the same codon as the original mutation.

ii. A nucleotide in a different codon is altered (e.g., an insertion frameshift is

suppressed by a nearby deletion event).


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b. Intergenic suppressors occur in a different gene (the suppressor gene)from the original mutation. Many work by changing mRNA translation.

i. Each suppressor gene works on only one type of nonsense, missense or

frameshift mutation.ii. A given suppressor gene suppresses all mutations for which it is specific.

iii. Suppressor genes often encode tRNAs that recognize stop codons and insert anamino acid, preventing premature termination of translation.

(1) Full or partial function of the polypeptide may be restored.

(2) The effect depends on how compatible the substituted amino acid is with protein function.

iv. Nonsense suppressors fall into three classes, one for each stop codon (UAG,

UAA and UGA) (Figure 19.5).v. Typical tRNA suppressor mutations are in redundant tRNA genes, so the wild-

type tRNA activity is not lost.

vi. Nonsense suppression occurs by competition between release factors andsuppressor tRNAs.

(1) UAG and UGA suppressor tRNAs do well in competition with release factors.

(2) UAA suppressor tRNAs are only 15% efficient.

vii. Suppression by a tRNA occurs at all of its specific stop codons (e.g., UGA orUAG), not just the mutant one. This may produce read-through proteins, but theyare not as common as expected, possibly due to tandem stop codons (e.g.,UAGUGA).


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tRNA suppressor gene mechanism for nonsense mutation


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New mutations are categorized as

inducedorspontaneous

Induced mutationsare defined as those thatarise after purposeful treatment withmutagens, environmental agents that areknown to increase the rate of mutations

Spontaneous mutationsare those that arisein the absence of known mutagen

treatment. They account for the"background rate" of mutation and arepresumably the ultimate source of naturalgenetic variation that is seen in populations.


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Spontaneous Mutations

1. All types of point mutations can occur spontaneously, duringS, G1 and G2 phases of the cell cycle, or by the movement of

transposons.

2. The spontaneous mutation rate in eukaryotes is between 10-

4-to-10-6 per gene per generation, and in bacteria and phages

10-5

-to-10-7

/gene/generation.a. Genetic constitution of the organism affects its mutation

rate.

i. In Drosophila, males and females of the same strain

have similar mutation rates.

ii. Flies of different strains, however, may have different

mutation rates.

b. Many spontaneous errors are corrected by the cellular

repair systems, and so do not become fixed in DNA.


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Different types DNA replication errors

Wobble-pairing

T-G, C-A, A-G, T-C

Normal pairing typically occurs in the next round of

replication; frequency of mutants in F2 is .GT pairs are targets for correction by proofreading andother repair systems

Additions and deletions

DNA loops out on template strand, DNA polymerase skipsbases, and deletion occurs

DNA loops out on new strand, DNA polymerase addsuntemplated bases


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Mutation caused by mismatch wobble base pairing


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Addition and deletion by DNA looping-out


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Spontaneous chemical changes

Depurination

Common; A or G are removed and replaced with arandom base.

Deamination

Amino group is removed from a base (C U); if notreplaced U pairs with A in next round of replication(CG TA).

Prokaryote DNA contains small amounts of 5MC;deamination of 5MC produces T (CG TA).

Regions with high levels of 5MC are mutation hotspots.


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Deamination


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Induced mutations

Radiation (e.g., X-rays, UV)

Ionizing radiation breaks covalent bonds includingthose in DNA and is the leading cause of chromosomemutations

Ionizing radiation has a cumulative effect and kills cellsat high doses

UV (254-260 nm) causes purines and pyrimidines to

form abnormal dimer bonds and bulges in the DNAstrands


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Thymine dimers induced by UV light.


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Induced mutations: chemical mutagens

Base analogs

Similar to normal bases, incorporated into DNA duringreplication

Some cause mis-pairing (e.g., 5-bromouracil)

Not all are mutagenic


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Mutagenic efffects

of 5-bromouracil


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Mutagenic efffects of 5-bromouracil


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Induced mutations: Chemical mutagens

Base modifying agents, act at any stage of

the cell cycle:

Deaminating agents

Hydroxylating agents

Alkylating agents


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Base-modifying agents

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Base-modifying agents (cont.).

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Induced mutations: chemical mutagens

Intercalating agents:

Thin, plate-like hydrophobicmolecules insert themselvesbetween adjacent base-pairs,

Mutagenic intercalatingagents cause insertionsduring DNA replication.

Loss of intercalating agentcan result in deletion.

Examples: proflavin, ethidiumbromide


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DNA Repair Mechanisms

1. Both prokaryotes and eukaryotes have

enzyme-based DNA repair systems that

prevent mutations and even death from

DNA damage.

2. Repair systems are grouped by their repair

mechanisms. Some directly correct, while

others excise the damaged area and then

repair the gap.


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Direct Correction of Mutational Lesions

1. DNA polymerase proofreading corrects most of the incorrect nucleotide

insertions that occur during DNA synthesis, which stalls until the wrongnucleotide is replaced with a correct one.

a. The role of 3-to-5 exonuclease activity is illustrated by mutator mutations in E.

coli, which confer a much higher mutation rate on the cells that carry them.

b. The mutD gene, encoding the e subunit of DNA polymerase III, is an example.

Cells mutant in mutD are defective in proofreading.

2. UV-induced pyrimidine dimers are repaired using photoreactivation (light

repair).

a. Near UV light (320370 nm) activates photolyase (product of the phr gene) to

split the dimer.

b. Photolyases are found in prokaryotes and simple eukaryotes, but not in

humans.

3. Damage by alkylation (usually methyl or ethyl groups) can be removed by

specific DNA repair enzymes.

a. For example, O6-methylguanine methyltransferase (from the ada gene)

recognizes O6-methylguanine in DNA, and removes the methyl group.

b. Demethylation restores the base to its original form.


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Repair Involving Excision of Base Pairs

1. Another repair system, which does not require light, was discovered

in 1964. It is called dark repair, the excision repair system, or thenucleotide excision repair (NER) system.

a. In E. coli, NER corrects pyrimidine dimers and other damage-induced distortions

of the DNA helix.

b. The proteins required are UvrA, UvrB, UvrC and UvrD (encoded by genes of the

same name) (Figure 19.17).

c. A complex of two UvrA and one UvrB proteins slides along the DNA. When it

encounters a helix distortion, the UvrA subunits dissociate, and a UvrC binds the

UvrB at the lesion.

d. When UvrBC forms, the UvrC cuts 45 nucleotides from the lesion on the 3 side,

and eight nucleotides away on the 5 side. Then UvrB is released and UvrD binds

the 5 cut end.e. UvrD is a helicase that unwinds the region between the cuts, releasing the short

ssDNA, while DNA polymerase I fills the gap and DNA ligase seals the

backbone.

f. In yeast and mammalian systems, about 12 genes encode proteins involved in

excision repair.


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Nucleotide excision repair (NER) of pyrimidine dimmer

and other damage-induced distortions of DNA


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2. Methyl-directed mismatch repair recognizes mismatched base

pairs, excises the incorrect bases and then carries out repair

synthesis.

a. In E. coli, initial stages involve products of the mutS, mutL and mutHgenes.

i. MutS binds the mismatch, and determines which is the new strand by its

lack of methylation.

ii. MutL and MutH bind unmethylated GATC sequences (site of methylation

in E. coli) and bring the GATC close to the mismatch by binding MutS.

iii. MutH then nicks the unmethylated GATC site, the mismatch is removedby an exonuclease and the gap is repaired by DNA polymerase III and

ligase.

b. Eukaryotes also have mismatch repair, but it is not clear how old and new

DNA strands are identified.

i. Four genes are involved in humans, hMSH2 (homologous to E. coli

mutS), and hMLH1, hPMS1 and hPMS2(all homologous to mutL).ii. All of these are mutator genes, and mutation in any 1 of them confers

hereditary predisposition to hereditary nonpolyposis colon cancer(HNPCC).


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Mechanism of mismatch correction repair


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3. The SOS response in bacteria results when specific base pairing

cannot occur. It allows the cell to survive otherwise lethal events, but

usually at the cost of incurring new mutations.

a. E. coliSOS is controlled by two genes, lexA and recA. (Mutants in either

of these genes have their SOS response permanently turned on.)

i. When no DNA damage is present, LexA represses transcription of

about 17 genes with products involved in various types of DNA

repair.

ii. Sufficient DNA damage activates the RecA protein, which

stimulates LexA to autocleave, removing repression of the DNA

repair genes.

iii. After damage is repaired, RecA is inactivated, and newly

synthesized LexA again represses the DNA repair genes.

b. The SOS system is an error-prone bypass synthesis system.

i. Some lesions, like T^T dimers, are easily copied to give AA in the

new DNA strand.

ii. Others, like C^C dimers, stall the SOS repair system, creating a

delay during which C can be deaminated to a U (forming a C^U),

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mutation 4th

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