Mutation

Mutation types

• Alteração na sequência nucleotídica• Várias classsificações

– Tipo de célula: somática ou linha germinal– Tipo de alteração molecular– Efeito fenotípico (na função)– Origem:

• espontâneas • induzidas

– Agentes químicos (mutagéneos)– Agentes físicos

• Sistemas de reparação

ACERCA DE MUTAÇÕES…

Mutation might also occur during DNA replication

Wilde-type

Mutant

Two basic classes of mutations: somatic andgerm-line mutation

Mosaics

Reproductive cells

Nonreproductive 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

Base substitution during replication leads to two types of molecular change:- transition- tranversion

Codons that can result from a single base change intyrosine codon UAU

Normalprotein

Normalprotein

Incompleteprotein

Unpredictbleprotein function

Different types of mutations caused bybase substitutions in coding regions

Ponctual mutations

Molecular basis of sickle-cell anemia. Consequences ofbase substitution example- missense mutation

The resulting hemoglobin is defective and tends to polymerize at low oxygen concentration

Insertion/deletion of a nucleotide

Frameshift mutation

Other classifications for phenotypic effects ofmutations

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

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

• Hypomorphic (leaky) (reduces normal function, usuallydue to low level gene expression)

• Hypermorphic (increases normal function, usually dueto high level gene expression)

Terminologia de mutação(frequentemente aplicada a microorganismos)

• Auxotrofos- não cresce em meio mínimo porque a mutação afecta um gene que codifica uma molécula biológica essencial

• Constituitivos- expressão (transcrição) permanente de um gene. Ex. região do operador do operão lac

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

• 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 thesame gene that contains the mutation beingsupressed

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

An INTERGENIC supressor mutation occurs in a different genefrom 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– Nonsense– Read-through– Frameshift

• 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)

vsInduced mutation

(in presence of known mutagen)

Spontaneous chemical changes

• Tautomerization

• Depurination

• Deamination (may also be induced by mutagenicchemicals)

iminoamino

keto enol

(tautomeric forms)

keto enol

amino imino

=NH

C-OH

-NH2

C=O

Pairing relationships of DNA bases in the normal and tautomericforms

(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

Desaminação

Perda do grupo amina NH2

Espontânea ou induzida

Deamination: spontaneous loss ofamino group

Methylated cytosine

Methylation of cytosine at the number-5 position in thebase. The methyl donor is S-adenosylmethyonine

Mechanism by which uracil-containing nucleotidesare 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 backboneby 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)G – C (wt)

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)– Hydroxylamine

• Intercalating agents (EtBr, proflavin …)• Reactive forms of oxygen (ex superoxide radicals)- oxidative

reactions

Base analogues(ex)

Principal mechanism of mutagenesis of base analogs: increased rate on base mispairing

Mispairing mutagenesis by 5-bromouracil

Normal pairing Mispairing

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 theposition at which the nucleotide is modified and the type

of of alkyl group that is added.Alkylation may alter

base-pairing propertiesand so lead to point mutations,

or cause structure distortion forming crosslinks between the two strands, blocking replication.Principal mechanism of mutagenesis: bulky attachments made to side groups on bases

Adenine deamination due to nitrous acidtreatment

Altered A pairs with C

Transition

A-T G-C

Pu-Py Pu-Py

INTERCALATING AGENTS

Insert between adjacent bases in DNA, distorting the three-dimensionalstructure of the helix and causing single-nucleotide insertions anddeletions in replicationreplication

Physical agents

UVIonizing radiation

Heat

In the electromagnetic spectrum, as wavelenght decreases, energy increases

Ionizing radiation

sunlight

/TV

Distortion of the DNA helixDNA replication and transcription are blocked

Pyrimidime dimers result from ultraviolet light

Different types of bonds betweenthe thymine rings are also possible

Ionizing radiation

• Source: x-rays, radon gas, radioactivematerials

• Mechanism of mutagenesis: – single and double-stranded breaks in DNA– damage to nucleotides

Técnicas de Mutagénese

Aleatória (random)Dirigida

Chemical mutagenesis using sodium bisulfite

Transição: C-G T-A

deamination

Oligonucleotide-directed mutagenesis by enzymatic primer extension

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

Enrichment for oligonucleotide-directedmutants by using a uracil-containing template

Single-stranded DNA is prepared in a ung- dut-

E. coli strain

Following ligation, the heteroduplex DNA moleculesare introduced in a ung+ E. coli strain

ung– - DNA uracil glycosylase deficientdut– - dUTPAse deficient (high levels of dUTP)

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 (não substituem o nt alterado, mas repõem a sua estrutura original)

– 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

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

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

– Recombinção homóloga em gaps ou cortes em cadeia dupla

Sistemas de reparação (cont.)

• Excisão– Excisão de bases e nts

• Glicosilases (enzimas específicas de re+aração do DNA). Ex. uracilglicosilase (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 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 cidoa ligação de grupos químicos)- Sistema UvrABC

• Sistema SOS (E. coli)

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

Direct repair: enzymatic removalchanges nucleotides back into their original stuctures

- ADA in E.coli- MGMT (O6-methylguanine-DNA methyltransferase)in humans

Base and nucleotide excision repairExcises modified bases and thenreplaces the entire nucleotide

Each DNA glycosylase enzymerecognizes and removes a specifictype of damaged base, producingan apurinic or an apyrimidinic site (AP site)

The endonuclease AP cleavesthe phosphodiester bond onthe 5’ side of the AP site andremoves the deoxyribose sugar

Gap

Nick

Many incorrectly inserted nucleotides thatescape proofreading are corrected byMutHLS - mismatch repair

Helicase and single-stranded exonuclease remove nucleotideson yhe new strand between theGATC sequence and the mismatch

Just after DNA replication…

The mismatch is brought close to a methylated GATC sequence, andthe new strand is identified

DNA polymerase I, DNA ligase

Dam methylase

Excision repair of DNA by E. coli UvrABC mechanism

UvrA/UvrB complex detect conformational changes in DNA

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 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