MUTATIONS

• Defn: Permanent chagnes in nucleotide sequence.

• occur in somatic cells or germline cells.• only germline cells inherited.

• somatic mutations believed responsible for many medical problems

• many cancers?

• Heritable change in the nucleotide sequence of a chromosome.

• Mutations can arise through exposure to mutagenic agents but the vast majority occur spontaneously through errors in DNA replication and repair.

• Although mutations can occur either in non-coding or coding sequences, it is only when they occur in the latter that they are recognised as an inherited disorder or disease.

• A mutation arising in a somatic cell cannot be transmitted to offspring, whereas if it occurs in gonadal tissue or a gamete it can be transmitted to future generations.

TYPES OF MUTATIONS

• mutations can be considered in two main classes according to how they are transmitted.

• mutations had been considered to be transmitted unaltered as what are termed fixed or stable mutations.

• More recently a new class of mutations, known as dynamic or unstable mutations, have been demonstrated to undergo further alteration as they are transmitted in families.

A- Fixed/stable mutations

• Fixed/stable point mutations can be classified according to the specific molecular changes at the DNA level.

• These include single base pair substitutions, insertions, deletions or duplications of part of a gene or DNA sequence.

1- Substitutions:

• A substitution is the replacement of a single nucleotide by another.

• If the substitution involves replacement by the same type of nucleotide, i.e. a pyrimidine for a pyrimidine (C for T or vice versa) or a purine for a purine (A for G or vice versa), this is termed a transition.

• Substitution of a pyrimidine by a purine or vice versa is termed a transversion.

Nucleotide Misincorporation

-C-A-G-C-T-

-G-T-C-G-A-

-C-A-G-C-T-

-G-T-C-G-A-

-C-A-G-C-T-

-G-T-T-G-A-

-C-A-G-C-T-

-G-T-C-G-A-correctly copied

CT substitution

• A deletion involves the loss of one or more nucleotides.

• If it occurs in coding sequences and involves one, two or more nucleotides which are not a multiple of three, it will disrupt the reading frame.

2- Deletions:

3- Insertions:

• An insertion involves the addition of one or more nucleotides into a gene.

• Again, if an insertion occurs in a coding sequence and involves one, two or more nucleotides which are not a multiple of three, it will disrupt the reading frame.

Added Nucleotides

-C-A-G-C-T-

-G-T-C-G-A-

-C-A-G-C-T-

-G-T-C-G-A-

-C-A-G-C-T-

-G-T-C G-A-

-C-A-G-C-T-

-G-T-C-G-A-

A

correctly copied

nucleotide added

B- Dynamic/unstable mutations

• unstable or dynamic mutations consist of triplet repeat sequences which, in affected persons, occur in increased copy number when compared to the general population.

• triplet amplification or expansion has been identified as the mutational basis for a number of different single gene disorders.

• The mechanism by which amplification or expansion of the triplet repeat sequence occurs is not clear at present.

Diseases arising from triplet repeat expansion

Disease Repeat sequence Repeat number Mutation number Repeat location

Huntington's disease (HD) CAG 9-35 37-100 Coding

Mvotonic dystrophy (DM) CTG 5-35 50-4000 3' UTR

Fragile X site A (FRAXA) CGG 10-50 200-2000 5' UTR

Kennedy disease (SBMA) CAG 17-24 40-55 Coding

Spino-cerebellar ataxia I-(SCAT) CAG 19-36 43-81 Coding

Spino-cerebellar ataxia 2 (SCA2) CAG 15-24 35-39 Coding

Machado-Joseph disease (MJD, SCA3) CAG 12-36 67->79 Coding

Spino-oaebellar ataxia 6 (SCA6) CAG 4-16 21-27 Coding

Spuio-ce ebellar ataxia 7 (SCAT) CAG 7-35 37-200 Coding

Spmo-oaebellar ataxia 8 (SCA8) CTG 16-37 100->500 UTR

I tatorubral-pallidoluysian atrophy (DRPLA) CAG 7-23 49->75 Coding

Friedreich's ataxia (FA) GAA 17-22 200-900 Intronic

Fragile X site E (FRAXE) CCG 6-25 >200 Promoter

Fragile X site F (FRAXF) GCC 6-29 >500 ?

Fragile 16 site A (FRA16A) CCG 16-49 1000-2000 ?

UTR = untranslated region.

Triplet Expansion in Fragile X

(CCG)n

FMR-1

200

60

6

No.triplets

Affected

Carrier

Normal

5’ 3’

DNA

STRUCTURAL EFFECTS OF MUTATIONS ON THE PROTEIN

• Mutations can also be subdivided into two main groups according to the effect on the polypeptide sequence of the encoded protein, being either synonymous or non- synonymous.

A- Synonymous/silent mutations

• If a mutation does not alter the polypeptide product of the gene, this is termed a synonymous or silent mutation.

• A single base pair substitution, particularly if it occurs in the third position of a codon, will often result in another triplet which codes for the same amino acid with no alteration in the properties of the resulting protein.

B- Non-synonymous mutations

• If a mutation leads to an alteration in the encoded polypeptide, it is known as a non-synonymous mutation.

• Alteration of the amino acid sequence of the protein product of a gene is likely to result in abnormal function.

• Non-synonymous mutations can occur in one of three main ways.

1- Missense

• A single bp substitution can result in coding for a different amino acid and the synthesis of an altered protein, a so-called missense mutation.

MIS-SENSE MUTATIONe.g. Sickle Cell Anaemia

Cause: defective haemoglobin due to mutation in β-globin geneSymptoms: severe anaemia and death in homozygote

Normal β-globin - 146 amino acids

val - his - leu - thr - pro - glu - glu - --------- 1 2 3 4 5 6 7

Normal gene (aa 6) Mutant gene

DNA CTC CAC

mRNA GAG GUG

Product Glu Valine

Mutant β-globin

val - his - leu - thr - pro - val - glu - ---------

2- Nonsense

• A substitution which leads to the generation of one of the stop codons will result in premature termination of translation of a peptide chain or what is termed a nonsense mutation.

1.Types of Mutationa) Substitution

Codon amino acid

Missense GAG glu

AAG lys

Nonsense GAG glu

Change to termination UAGstop

Silent GAG glu

GAA glu

3- Frameshift

If a mutation involves the insertion or deletion of nucleotides which are not a multiple of three, it will disrupt the reading frame and constitute what is known as a frameshift mutation.

Frameshift

RNA AUG UGU UAU CAU UGG

Insertion AUG UUG UUA UCA UUG G

Deletion AUG UGU UUC AUU GG

ANote amino acids translated

Mutations in non-coding DNA

• Mutations in non-coding DNA, in general, would not be expected to have a phenotypic effect unless they occur in the DNA sequences involved in gene regulation, e.g. the TATA box, or in the splicing of introns, e.g. the highly conserved GT and AG dinucleotides at the end of introns.

• Mutations in regulatory elements can affect the level of gene expression while mutations in splice junctions can result in coding sequences being lost or intronic sequences being added to the mRNA molecule.

FUNCTIONAL EFFECTS OF MUTATIONS ON THE PROTEIN

• The mutations effect can appear either through loss- or gain-of-function.

1- Loss-of-function:

• These mutations can result in either reduced activity or complete loss of the gene product.

• Loss-of-function mutations in the heterozygous state would be associated with half normal levels of the protein product.

2- Gain-of-function mutations

• Gain-of-function mutations, as the name suggests, result in either increased levels of gene expression or the development of a new function(s) of the gene product.

THE END

MUTAGENS AND MUTAGENESIS

• Naturally occurring mutations are referred to as spontaneous mutations and are thought to arise through chance errors in chromosomal division or DNA replication.

• Environmental agents which cause mutations are known as mutagens.

MUTAGENS

• These include natural or artificial ionizing radiation and chemical or physical mutagens.

• Ionizing radiation includes electromagnetic waves of very short wavelength (X-rays and gamma rays), and high energy particles (α particles, ß particles and neutrons).

Causes of Mutations• radiation (nuclear accident

or x-ray)

• chemicals

Mutagenesis

“Process of producing a mutant”

spontaneous induced

Mutagen: physical agent or a chemical which causes or increases the rate of mutation

Causes of Spontaneous mutations

• error in DNA replication

• faulty DNA repair e.g. mis-match

• bases form tautomers which pair differently

• natural mutagens

DNA Mismatch Repair

A G C T GA G C T G

TT CC TT AA CC

T C G A CT C G A C

A G C T GA G C T G

A G C T GA G C T G

TT CC TT AA CC

Base pair Base pair mismatchmismatch

Normal DNA Normal DNA repairrepair

Mutation Mutation introduced by introduced by

unrepaired DNAunrepaired DNA

T C T C TT A C A C

A G A G AA T GT G

Mutagens

PHYSICAL CHEMICAL BIOLOGICAL

USE ResearchDiseaseWarfare

Physical Mutagens

Radiation

• Non-ionising

– Ultraviolet (UV)

• Ionising

– X-Rays

Mutagenic chemicals in food contribute to 35% of cancers!

THE END

Biological Mutagens

Insertion of a transposon within a gene

• disrupts the reading frame

• loss of function

Jumping genes- transposable elements move to different positions in the chromosome. Transposon carries other genes with it.

• (Antibiotic resistance)

1. UV (260nm)

Absorbed by bases of DNA and RNA• forms pyrimidine dimers• 2 adjacent bases (CC or TT) are covalently

joined• insertion of incorrect nucleotide at this point

likely• during replication Skin cancer, sterilisation of

equipment

Poor penetration

2. Ionising radiation

X-rays, cosmic rays, gamma rays

More powerful than UV

Penetrates glass

Water etc ionises: mutagenic effect

• free radicals e.g. OH-

• inactivate DNA

• cell death

Chemical Mutagens

1.Base analogues

Resemble DNA bases

Faulty pairing properties

Example 5-bromouracil

• incorporated like T

• faulty pairing with G