Mutation
Mutation types
ACERCA DE MUTAÇÕES…
• Alteração na sequência nucleotídica
• Várias classsificações
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–
–
–
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
Mutation might also occur during DNA replication
Wilde-type
Mutant
Two basic classes of mutations: somatic and
germ-line mutation
Nonreproductive cells
Reproductive cells
Mosaics
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 in
tyrosine codon UAU
Different types of mutations caused by
base substitutions in coding regions
Normal
protein
Ponctual mutations
Unpredictble
protein function
Incomplete
protein
Normal
protein
Molecular basis of sickle-cell anemia. Consequences of
base 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 of
mutations
• Loss-of-function (null or knockout) (eliminates normal
function)
• Gain-of-function (ectopic expression) (expressed at
incorrect time, or in appropriate cell types)
• Hypomorphic (leaky) (reduces normal function, usually
due to low level gene expression)
• Hypermorphic (increases normal function, usually due
to 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 the
same gene that contains the mutation being
supressed
Model for the effect of mutation
and intragenic supression on
the folding and activity
An INTERGENIC supressor mutation occurs in a different gene
from the one bearing the original mutation
Mutation in a different gene
Leu tRNA gene
Mutant Leu tRNA gene
X
Mutant Leu tRNA
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
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–
–
–
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
Induced mutation
(in presence of known mutagen)
Spontaneous chemical changes
• Tautomerization
• Depurination
• Deamination (may also be induced by mutagenic
chemicals)
(tautomeric forms)
keto
enol
C=O
C-OH
keto
enol
amino
imino
-NH2
=NH
amino
imino
Pairing relationships of DNA bases in the normal and tautomeric
forms
(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 of
amino group
Methylated cytosine
Methylation of cytosine at the number-5 position in the
base. The methyl donor is S-adenosylmethyonine
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)
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 the
position at which the nucleotide is modified and the type
of of alkyl group that is added.
Alkylation may alter
base-pairing properties
and 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 acid
treatment
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-dimensional
structure of the helix and causing single-nucleotide insertions and
deletions in replication
Physical agents
UV
Ionizing radiation
Heat
In the electromagnetic spectrum, as wavelenght decreases, energy increases
Ionizing radiation
sunlight
/TV
Pyrimidime dimers result from ultraviolet light
Different types of bonds between
the thymine rings are also possible
Distortion of the DNA helix
DNA replication and transcription are blocked
Ionizing radiation
• Source: x-rays, radon gas, radioactive
materials
• Mechanism of mutagenesis:
– single and double-stranded breaks in DNA
– damage to nucleotides
Técnicas de Mutagénese
Aleatória (random)
Dirigida
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 colonies
and is screened to identify mutants
Enrichment for oligonucleotide-directed
mutants by using a uracil-containing template
Single-stranded DNA is prepared in a ung- dutE. coli strain
ung– - DNA uracil glycosylase deficient
dut– - dUTPAse deficient (high levels of dUTP)
Following ligation, the heteroduplex DNA molecules
are introduced in a ung+ E. coli strain
Quick-Change site directed mutagenesis
DNA isolated from most E. coli strains
is dam methylated
DpnI- is specific for methylated and
hemimethylated DNA
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. 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
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 + …)
Direct repair: enzymatic removal
changes nucleotides back into their original stuctures
- ADA in E.coli
- MGMT (O6-methylguanine-DNA methyltransferase)
in humans
Base and nucleotide excision repair
Excises modified bases and then
replaces the entire nucleotide
Each DNA glycosylase enzyme
recognizes 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
removes the deoxyribose sugar
Gap
Nick
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
Many incorrectly inserted nucleotides that
escape proofreading are corrected by
MutHLS - mismatch repair
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