Mechanisms of Gene Mutation 2 Flashcards
Spontaneous Mutations:
- DEPURINATION
- DEAMINATION AND
- OXIDATIVE DAMAGE.
Naturally occurring DNA damage is rare.
Spontaneous lesions generate mutations:
- Depurination
- Deamination (eg smoked meat and preservatives -nitrous acid)
- Oxidative damage (eg free radicals)
Understanding spontaneous mutations: DEPURINATION
Depurination: loss of a purine.
- In vitro Mammalian cells can lose 10 000 purines from DNA in 20h cell-generation period at 37C.
- In vivo some loss and If such lesions persist, would cause significant genetic damage, as apurinic sites can’t specify a base pair during replication
BUT, a base can be inserted opposite an apurinic site, often causing a mutation (USUALLY THEREFORE A TRANSVERSION).
Understanding Depurination and Depyrimidination
A. Fox will cover, 99% of time repaired
- Create AP sites (apyrimidinic or apurinic)
- No base for pairing during DNA replication
- Can lead to insertion of a
base without pairing - (SOS response): inserted
base can be wrong - MUTATION
or
1. Create AP sites (apyrimidinic or apurinic)
- REPAIR
Understanding spontaneous mutations: Deamination
Deamination: Sodium nitrate (NaNO3) common food additive, also occurs naturally in meats preserved by smoking.
In the stomach NaNO3 is converted to Nitrous Acid (HNO2) which acts as a mutagen by deamination.
Original base: cytosine
mutagen: nitrous acid (HNO2) –DEAMINATION
MODIFIED BASE: uracil
PAIRING PARTNER: adenine
CG to TA
Uracil Glycosylase
If uracil is in DNA
—DNA glycosylase removes base
- Usually formed by cytosine deamination to Uracil
- Removes uracil
- Leaves an AP site
- Repair or mutation (More often, 99.9% repaired)
If uracil is in DNA
—DNA glycosylase removes base STEPS 5
- Each DNA glyosylase recognises and removes a specific type of damaged base, producing an apurinic or an apyrimidinic site (AP site)
- AP endonuclease cleaves the phosphodiester bond on the 5’side of the AP site…
- …and removes the deoxyribose sugar
- DNA polymerase adds new nucleotides to be exposed 3’-OH group
- The nick in the sugar-phosphate backbone is sealed by DNA ligase, restoring the original sequence.
Deamination
5-Methylcytosine (5mC) —> deamination (NH2) —> Thymine
(Many eukaryotes contain methylated cytosine)
Mutation frequency at sites of 5-methylcytosine is higher than at cytosine
Deamination: of C yields U. If not corrected, U will pair
with A and give a GC to AT transition.
Deamination: of 5-methyl-C yields T, so will get a GC to
AT transition. In E. Coli lac1 gene hot spots for such
transitions have 5-methyl-C* at each hot spot.
Spontaneous Mutations
Oxidative damage:
Oxidative damage: Active oxygen species produced
as byproducts of normal aerobic metabolism.
superoxide radical (O2-) hydrogen peroxide (H2O2)
hydroxyl radicals (.OH-).
Cause damage to DNA - lead to mutations or replication
blocks, disease.
thymidine glycol
-Blocks replication, not shown to cause mutations (apoptosis)
8-Oxo-7-hydrodeoxyguanosine
- 8-oxo dG
mispairs with
A = G to T
transversion
UnderstandingUnequal Crossing Over - indel mutations = 3
- if homologous chromosomes misalign during crossing over,…
- …one crossover product contains an insertion …
- …and the other has a deletion.
**Frequently the cause of colour blindness in humans
Errors in DNA Replication
Mistakes made by DNA replication machinery are
another source of mutation:
- Base substitutions
- Base insertion/deletion
understanding base substitutions
Base substitutions: No chemical reaction is 100%
efficient, hence can get illegitimate base pairing
during DNA replication.
Recall: Tautomers and wobble base pairs also can cause mispairing.
understanding ase insertion/deletion:
Base insertion/deletion: Replication errors can lead
to indel mutations. If the number of base pairs is
not a factor of 3, then get frameshift. Such indel
mutations often occur at repeated bases.
Replication Error - Strand Slippage
**Know how replication slippage can impact the number of nucleotides!
pg 45
- Newly synthesised strand loops out,..
- …resulting in the addition of one nucleotide on the new strand.
- template strand loops out,…
- …resulting in the omission of one nucleotide on the new strand.
understanding Trinucleotide Repeat Diseases
Common mechanism responsible for a number of genetic diseases in humans is expansion of a 3bp repeat - hence the name!
STEPS
1. Trinucleotide repeats (CAGn)
2. Slipped mispairing
– STRAND SLIPS DURING DNA REPLICATION
- Part of the template is now repeated 2x in the daughter strand
- 4 triplets (12nts) looped out
(GTCn)
The trinucleotide repeat might be within or outside of the protein coding region…
The trinucleotide repeat might be within or outside of
the protein coding region…
Examples of human genetic diseases caused by expanding nucleotide repeats
Fragile X syndrome
REPEATING SEQUENCE: CGG
Normal range :6-54
Disease range: 50-1500
HUNTINGTONS DISEASE
CAG
9-37
37-121
Spinal and bulbar muscular atrophy
JACOBSEN SYNDROME
AUTOSOMAL DOMINANT CEREBELLAR ATAXIA
SPINOCEREBELLAR ATAXIA (sev. types)
Myotonic syndrome
friedreich ataxia
dentatorubal-pallidoluysian atropy
myclonus epilepsy of the unverricht-lundborg type
Trinucleotide Repeat Diseases
Eg, Fragile X syndrome - the most common form of inherited mental retardation (1 in 1500 males, 1 in 2500
females).
Seen cytologically by a fragile
site on X-chromosome, causing breaks in vitro.
FRAGILE X RESULTS FROM CHANGE IN NUMBER OF (CGG)n REPEATS IN A REGION OF THE FRMR-1 GENE that is transcribed, but not translated
(EXON 1 BEFORE THE AUG START CODON SO NOT PART OF THE PROTEIN)
Fragile X Syndrome
- Usually have variation between people in number of CGG repeats in FMR-1 gene (6 - 54 repeats, most frequent allele contains 29 repeats).
Unaffected parents & grandparents
(with 50 to 200 repeats) = premutation,
have offspring with
fragile X - have 200 to 1300 repeats..
CpG island -> hypermethylation
Fragile X Syndrome SUMMARY
Unaffected parents & grandparents (with 50 to 200 repeats) = premutation
- not sufficient to cause disease, but are readily expanded (not stable) cf normal alleles.
Expansion caused by slipped mispairing during DNA replication.
It seems that in
human cells after 50 or so repeats, the replication machinery can’t faithfully copy DNA, resulting in large variations in repeat
number.
Huntington Disease SUMMARY
Late onset (mid to late 30s), dominant disorder resulting in neural degeneration (so likely pass onto children).
Also associated with expansion of trinucleotide (CAG) repeats (WITHIN CODING REGION).
**Severity of disease correlates with number of repeats. Normal person has 19 to 21 repeats,
Huntington patient has 40-60.
CAG encodes for Glu POLY GLUTAMINE EXPANSION CAUSES TOXICITY
Ames Test for Mutagenicity
Mutagens:
What type of mutations do they induce?
Test for…
Developed by
Mutagens: increase the frequency of mutation
What type of mutations do they induce?
Auxotrophic histidine mutants of Salmonella
Test for reversion to prototrophy in presence of
chemical agent (mutagen?)
Developed by Bruce Ames
Selection on Minimal Medium- ames test for mutagency
Tests for mutagenicity
of compounds in
bacterial model.
Many mutagens are
also carcinogens, some carcinogens are not themselves carcinogenic, but their metabolites are…
Ames Test for Aflatoxin B1
Need base substitution for
reversion to prototroph, eg
GC to TA transversions
Need indel mutations for
reversion to prototroph
Sequencing of DNA after Cell Exposure to
Mutagens
pg 55
understanding mutagenic specificity
For induction of mutations, choose a mutagen that
causes the type of changes that are useful
- base substitution,
- deletions and insertions,
- (frameshift mutation)
eg intercalators
Can assess whether an environmental mutagen is responsible for observed mutations
eg Aflatoxin B1 (GC to TA)
summary of entire lecture
- Mispairing & damage to nucleotides can occur spontaneously increase mutation
- Mutagens increase the rate of DNA changes by:
- damaging a base
- altering a base so it mispairs
- taking the place of a base
Ames test measures mutagenicity
