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
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