Mutation

Mutation types

• Alteração na sequência nucleotídica

• Várias classsificações

– Tipo de célula: somática ou linha germinal

– Tipo de alteração: molecular ou cromossómica

– Efeito fenotípico (na função)

– Origem:

ACERCA de MUTAÇÕES…

– Origem:

• espontâneas

• induzidas

– Agentes químicos (mutagéneos)

– Agentes físicos

• Sistemas de reparação

Mutation might also occur during DNA replication

Wilde-type

Mutant

Two basic classes of mutations based on the mutated type of

cell: somatic and germ-line mutation

MosaicsNonreproductive cells

Reproductive cells

Three basic molecular types of gene mutations are base substitutions, insertions and deletions

Base substitution

leads to two types of molecular change: transition and tranversion

Codons that can result from a single base change in tyrosine

codon UAU

Base substitutions

cause different types of mutations in coding regions

Normalprotein

Normalprotein

Incompleteprotein

Unpredictbleprotein function

Ponctual mutations

Molecular basis of sickle-cell anemia. Consequences of base

substitution example- missense mutation

The resulting hemoglobins are defective and tend to polymerize at low oxygen concentration

Insertion/deletion of a nucleotide

cause frameshift mutations in coding regions

Frameshift mutation(ex.)

Classifications for phenotypic effects of mutations

• Loss-of-function (null or knockout) - eliminates normal function

• Gain-of-function- expressed at incorrect time, or in appropriate cell types (ectopic expression)

• Haploinsufficiency- a form of dominance in which na individual heterozygous for a wild-type allele and null allele shows na abnormal heterozygous for a wild-type allele and null allele shows na abnormal phenotype because the level of gene activity is not enough to produce a normal phenotype

• Hypomorphic (leaky)- reduces normal function, usually due to low level gene expression

• Hypermorphic- increases normal function, usually due to high level gene expression

• Auxotrofos- não crescem em meio mínimo porque a mutação afecta um gene que codifica uma molécula biológica essencial (aplica-se a microorganismos)

• Constitutivos- expressão permanente de um gene, independentemente das condições (externas).

• Condicionais- só se manifestam sob determinadas condições (não permissivas). Ex. mutações termosensíveis (Ts)

• Letais condicionais- mutações que só se manifestam sob determinadas condições, e quando tal são letais

• Incondicionais- manifestam-se sob condições permissivas e não permissivas

Relation of foward, reverse and supressor mutations

An INTRAGENIC supressor mutation occurs in the same gene

that contains the mutation being supressed

Same amino acid

as in wt

Model for the effect of mutation and intragenic supression onthe folding and activity

Tyr- polar uncharged;

Gly- nonpolar;

Glu- hidrofílico c/ carga negativa;

Cys- nonpolar

Different amino acid

but the protein conformation,

though the protein fucntion, is reverted

An INTERGENIC supressor mutation occurs in a different gene

from the one bearing the original mutation

Leu tRNA gene

Mutant Leu tRNA geneX

Mutant Leu tRNA

Mutation in a different gene

Efeitos das mutações

• Mutações silenciosas– No DNA intergénico

– Em regiões não codificantes

– Numa base do tripleto, sem alterar o aa

• Mutações em regiões codificantes– Silenciosas

– Missense– Missense

– Nonsense

– Frameshift

– Read-through

• Mutações em regiões não codificantes, mas não silenciosas– Região do promotor

– Sequências reguladoras

– Origem replicação

– Limiar exão/intrão ou mesmo no intrão• Novos locis de splicing alternativo

Spontaneous mutation(in absence of known mutagen)

vs vs

Induced mutation(in presence of known mutagen)

Spontaneous chemical changes

• Tautomerization

• Depurination

• Deamination (may also be induced by mutagenic chemicals)

keto enol

(tautomeric forms)

keto enol

C-OHC=O

iminoamino

amino imino

-NH2 =NH

Pairing relationships of DNA basesin the normal andtautomeric forms

Tautomerization

(imino)

(enol)

Tautomeric shifts results in transition mutations. The tautomerization can occur in the:

- template base, ie, tautomerization of the base in the template

- substrate base, ie, tautomerization of incoming base.

Depurination

Spontaneous or inducedloss of amino group (NH2)Deamination

Memorandum:

Methylation of cytosine at the position 5 in the base

The methyl donor is S-adenosylmethyonine

Chemical induced mutations

Chemical environmental agents that

significantly increase the

rate of mutation above the spontaneous rate

Ex.

• Base analogs (ex. 5-Bu, 2-AP)

• Chemicals that alter bases– Nitrous acid- deamination

– Alkylating agents (EMS, NTG, nitrogen mustards, mitomycin C)

– Hydroxylating agents (hydroxylamine)

• Intercalating agents (EtBr, proflavin …)

• Reactive forms of oxygen (ex superoxide radicals)- oxidative reactions

Principal mechanism of mutagenesis of base analogs: increased rate on base mispairingex. mispairing mutagenesis by 5-bromouracil

Normal pairing Mispairing

A-T G-C substitution

During subsequent rounds

of replication different

bases are aligned opposite

the 5-BrU residue.

Base analogs ≠ Nucleotide analogue

AZT is used in the clinical treatment of AIDS

Chemicals may alter DNA bases

Highly mutagenic alkylating agents

The effect of alkylation depends on the

position at which the nucleotide/base pair is modified and the type of alkyl group that is added.

Alkylation may alter

base-pairing propertiesand so lead to

point mutations, or cause structure distortionforming crosslinks between the two strands, blocking replication.

Principal mechanism of mutagenesis: bulky attachments made to side groups on bases

Adenine deamination due to nitrous acid treatment

Altered A pairs with C

Transition

A-T G-C

INTERCALATING AGENTS

Insert between adjacent bases in DNA, distorting the three-dimensional structure of the helix and causing single-nucleotide insertions and deletions in replicationreplication

Physical agents

Ionizing radiationUltraviolet radiation

Heat

In the electromagnetic spectrum, as wavelenght decreases, energy increases

Ionizing radiation

sunlight

/TV

Ionizing radiation

• Source:

– x-rays, radon gas, radioactive materials

• Mechanism of mutagenesis: • Mechanism of mutagenesis:

– single and double-stranded breaks in DNA

– damage to nucleotides

Pyrimidime dimers result from ultraviolet light

Distortion of the DNA helix

DNA replication and transcription are blocked

Different types of bonds between

the thymine rings are also possible

Técnicas de Mutagénese

Aleatória (random)

Dirigida

Chemical mutagenesis

deamination

Chemical mutagenesis using sodium bisulfite

Transição: C-G T-A

Oligonucleotide-directed mutagenesis by enzymatic primer extension

Plasmid DNA is isolated from the resulting coloniesand is screened to identify mutants

Quick-Change site directed mutagenesis

DpnI- is specific for methylated and

hemimethylated DNA

DNA isolated from most E. coli strains

is dam methylated

Mutation Repair

Sistemas de reparação

• Directos

– Recombinção homóloga em gaps ou cortes em cadeia dupla

– DNA ligase que actua sobre cortes em cadeia simples (nicks)

DNA polimerase I e DNA ligase (E. coli) que actuam em lacunas – DNA polimerase I e DNA ligase (E. coli) que actuam em lacunas (gaps)

– Fotoreparação enzimática. Ex. fotoliase de E. coli

– Remoção enzimática de grupos químicos que se ligam às bases dos nts e os alteram. Ex enzima ADA de E. coli que remove os grupos alquilo na posição 6 da guanina

Direct repair: enzymatic removalchanges nucleotides back into their original stuctures

- ADA in E.coli

- MGMT (O6-methylguanine-DNA methyltransferase)

in humans

Sistemas de reparação (cont.)

• Excisão

– Excisão de bases e nts

• Glicosilases (enzimas específicas de reparação do DNA). Ex. DNA uracil glicosilase (ung). Geram locais apurínicos (Depurinação)

• Endonucleases AP- removem o açúcar-fosfato nos locais apurínicos (AP)

• Excisão de nucleótiodos pelo sistema MutHLS (geralmente associado a um • Excisão de nucleótiodos pelo sistema MutHLS (geralmente associado a um incorrecto emparelhamento de bases- mismatch)

• Excisão de nucleótidos devido a bases modificadas que distorcem a configuração normal do DNA. Ex. dímeros de timina, bases alteradas do cido a ligação de grupos químicos)- Sistema UvrABC

• Sistema SOS (E. coli)

• DNA clivado em ambas as cadeias (proteínas Ku70 e Ku80 + cinase de DNA + …)

Base and nucleotide excision repairExcises modified bases and then

replaces the entire nucleotide

Each DNA glycosylase enzymerecognizes and removes a specific

type of damaged base, producing

an apurinic or an apyrimidinic site

(AP site)

The endonuclease AP cleaves

the phosphodiester bond on

the 5’ side of the AP site and

Gap

the 5’ side of the AP site and

removes the deoxyribose sugar

Nick

Mechanism by which uracil-containing nucleotides are formed in DNA and removed (E. coli)

Uracil is cleaved from the deoxyribose sugar by DNA uracil glycosylase

The deoxyribose with the uracil detached is then excised from the DNA backbone

by another enzyme (AP endonuclease) and the gap is repaired

Deamination of 5-methylcytosine leads to a mutation

5MeC – G T – G Replication T – A (mutant)C – G (wt)

Transition

mutation

Many incorrectly inserted nucleotides that

escape proofreading are corrected by

MutHLS - mismatch repair or methyl-directed mismatch repair

Just after DNA replication…

The mismatch is brought close to a

methylated GATC sequence, and

the new strand is identified

Helicase and single-stranded exonuclease remove nucleotides

on yhe new strand between the

GATC sequence and the mismatch

DNA polymerase I, DNA ligase

Dam methylase

Excision repair of DNA by E. coli UvrABC mechanism

UvrA/UvrB complex detect

conformational changes in DNAEx. thymine dimers Helix to become locally denatured

and kinked by 130°

UvrC endonuclease binds and

cuts the damaged strand at two

sites separated by 12 or 13 basessites separated by 12 or 13 bases

Helicase II unwinds the damaged region,

releasing the single-stranded fragment

with the lesion, which is degraded to

mononucleotides

The gap is filled by DNA polymerase I, and

the remaining nick is sealed by DNA ligase