Download Chapter 10 Gene Mutation: Origins and Repair Processes

Chapter 10
Gene Mutation:
Origins and Repair Processes
Mutation is the process wherby genes change from
one allelic form to another.
Genetic analysis would not be possible without
mutants.
Gene mutations at the molecular level
Type of mutation
Examples
Forward mutations at DNA level
Transition
Purin replaced by different purin, or pyrimidine replaced
by a different pyrimidine: AT ⇒ GC; CG ⇒ TA
Transversion
Purine replaced by a pyrimidine, or pyrimidine replaced
by a purine: AT ⇒ CG; CG ⇒ GC
Forward mutations at protein level
Silent mutation
Triplets code for same amino acid: AGG ⇒ CGG (Arg)
Synonymous missence mutation
Codon specifies a chemically similar amino acid
Nonsynonymous missence mutation Codon specifies a chemically dissimilar amino acid
Type of mutation
Frameshift mutation
Reverse mutations
Exact reversion
Equivalent reversion
Examples
Any addition or deletion of base pairs that is not a
multiple of 3.
forward
AAA(Lys)
Wild-type
UCC(Ser)
Wild-type
CGC(Arg)
Wild-type
⇒
GAA(Glu)
Mutant
forward
⇒
UCG(Cys)
Mutant
forward
⇒
CCC(Pro)
Mutant
reverse
AAA(Lys)
Wild-type
⇒
reverse
⇒
AGC(Ser)
Wild-type
reverse
⇒
CAC(His)
Pseudo wild-type
Intragenic suppressor mutation
For example, compensation of a frameshift mutation by
a second mutation
Extragenic suppressor mutation
Nonsense suppressors, missense suppressors,
frameshift suppressors, physiological suppressors
Consequences of point mutations
Salvatoria Luria and Max Delbrück's fluctuation test
Hypothesis: random mutation or directed physiological change (1943)
The frequency of mutations can be enhanced by mutagend
Mechanism of point-mutation induction
a) Base replacement by base analogs
All bases can exist in one of several forms, called tautomers, which are
isomers that differ in the positions of their atoms and in the bonds
between the atoms.
Mutation by tautomeric shifts
Alternative pairing possibilities for 5-bromouracil. 5-BU is an analog of
thymine that can be incorporated into DNA. 5-BU switches relatively
frequently to the ionized form.
Mechanisms of mutation induction
b) Base alteration
Some mutagens alter a base, causing specific mispairing:
O
C2H5 O S CH3
O
Ethyl methanesulfonate
Mechanisms of mutation induction
c) intercalating agents
Intercalating agents inhibit DNA synthesis and cause small deletions or
insertions
Mechanisms of mutation induction
d) Base damage
Many mutagens damage bases with the consequence that no
specific base pairing is possible.
Examples: UV light, Aflatoxin B1.
The SOS system
DNA polymerase stops at a noncoding lesion, generating ssDNA that attracts SsB and RecA which
forms filaments. These filaments stimulate the expression of UmuD. UmuD is cleaved by RecA to
yield UmuD' and UmuC which permit the DNA polymerase to proceed with DNA synthesis.
Mutations are caused, because the inserted bases have a high error frequency.
UV light generates pyrimidine photo dimers.
A cyclobutan and a
6-4 photoproduct
can be formed.
These dimers
interfere with normal
base pairing.
Ames test
Cytochrom P450
monooxygenases in the liver
convert certain metabolites by
hydroxylation into mutagens.
Ames test showing the
mutagenicity of aflatoxin.
Salmonella strain TA100 is sensitive to
reversion through base substitution.
Strains TA1538, TA1535 are sensitive
to reversion by frameshift mutation.
Binding of metabolically activated aflatocin
Spontaneous Mutation
Spontaneous lesions are caused by
deamination or depurination.
DNA replication errors can also lead
to mutations.
Deamination of 5-methlyctosin generates a "normal" base
Damage products formed attack by oxygen radicals
dR: deoxyribose
Biological repair mechanisms
1.
2.
3.
4.
5.
Photolyase
Excision repair
Nucleotide exision repair
Mismatch repair
Post-replication repair
Photolyases catalyse the repair
of pyrimidine dimers.
The enzyme uses energy from
visible light to break the carboncarbon bonds that join adjacent
pyrimidine residues after UV
irradiation.
The base excision repair pathway.
In this example, a uracil that was formed by
deamination of cytosin is removed from the
sugar-phosphate backbone by DNA
glycosylase. These enzymes create apurinic or
apyrimidinic (AP) sites. AP endonuclease
recognizes these AP sites and cleaves the
DNA strand. The remaining deoxyribose
molecule is removed by deoxyribose
phosphodiesterase and the gap is filled.
Nucleotide excision repair (of a
pyrimidine dimer).
The DNA replication machinery is used
to remove distortions in the DNA
double helix. Damaged DNA is first
recognized by endonucleases and
cleaved. The oligonucleotide is then
exised after helicase unwindes the
DNA at this site.
Eukaryotic nucleotide excision
repair in the context of
chromatin.
Highly conserved among eukaryotes.
About 25 proteins are needed for the
ordered process of damage
recognition, excision of damaged
DNA and repair synthesis. For repair,
about 100bp of nucleosome free DNA
are required. Nucleotide excision
repair (NER) proteins are mobile. After
DNA damage, NER proteins assemble
transiently at the site of DNA lesion.
One repair event needs ca. 4 minutes.
After repair, the chromation assembly
factor CAF-1 restores the chromatin
structure.
Xeroderma pigmentosum, a human
disorder, results from a defect in any
one of eight genes involved in
nucleotide excision repair.
Mismatch repair in E. coli.
Mismatch repair corrects errors made
during DNA replication. A complex of
three proteins (MutH, MutL, MutS)
recognizes mismatched bases
introduced by DNA polymerase into the
newely synthesized, unmethylated DNA
strand. MutH cleaves the new strand
opposite the methylated site and the
damaged DNA is then removed.
Post-replication repair.
Extensive DNA damage can be repaired
by homologous recombination. An
unrepaired lesion can be bypassed by
DNA polymerase. The resulting gap is
then repaired by recombination with the
undamaged parental strand.
Mutational analysis
1. Somatic versus germinal mutation
2. Mutant sectors
3. Mutant phenotypes
Somatic versus germinal mutation
Mutant sectors
Summary