Mechanisms of Gene Mutation Flashcards
Mutagens are characterised by: … for ….
distinct mutational specificity
for both the type of
MUTATION & for the SITE of the mutation
What are “HOT SPOTS” for mutations?
“Hot spots” are sites in genes where MUTATIONS OCCUR MORE FREQUENTLY than other sites in the
gene;
may be spontaneous or induced mutation
Mechanisms of POINT MUTATION Induction
—Mutagens can act through at least three different
mechanisms:
- can REPLACE a base in the DNA
- can ALTER a base & cause it to MISPAIR
- can DAMAGE a base so it CAN’T PAIR
Mechanisms of Mutation Induction: 2 AND EXPLAIN
- Base mispairing
-TAUTOMERIC forms of bases
- IONISED bases
- PURINE - PURINE & PYRIMIDINE-PYRIMIDINE (less common)
2.Frameshift
- slipped mispairing (slippage during DNA rep)
- unequal crossing over (during recombination or
meiosis)
EXPLAIN BASE REPLACEMENT
Base replacement - BASE ANALOGUES are STRUCTURALLY SIMILAR to bases & may be inserted
into DNA.
However, base analogues often DO NOT PAIR CORRECTLY - hence can cause CAUSE MUTATIONS by insertion of incorrect nucleotide during replication
Explain BASE ALTERATIONS
Base alterations - bases can have different forms
= TAUTOMERS
Base tautomers are in equilibrium.
Keto (>C=O) form normally present in DNA.
Imino (>C=NH) & enol (>C-OH) forms rare.
Imino & enol base tautomers may pair with the wrong base = mispair,
Imino & enol base tautomers may pair with the wrong base = mispair,
Explain WOBBLE base Pairing…
- DNA strands separate for replication.
- Thymine on the original template strand base pairs with guanine through wobble, leading to an incorporated error.
- At next round of replication, the guanine nucleotide pairs with CYTOSINE, leading to a TRANSITION MUTATION.
What is INCORPORATED ERROR?
Thought to be responsible for many of the mispairings in DNA replication
Incorporated error = mispair in NEW strand… in the next
round of replication becomes a fixed/permanent replicated error (TA -> CG).
What type of mutation is this?
Mutations Caused by Base Mispairing:
- Typically transitions:
- purine to purine
- pyrimidine to pyrimidine
Due to wrong partners in purine - pyrimidine pair
G with T: GC to AT
A with C: AT to GC
2.Rarely transversions:
Purine to Pyrimidine
Pyrimidine to Purine
Due to purine-purine pairing or pyrimidine- pyrimidine pairing
G with A: GC to TA
T with C: TA to GC
Energetically unfavourable (distort dimensions of double helix)
Understanding Base Ionisation:
Mispairs can also be formed when base analogues
are ionised 5-bromouracil a thymidine analogue, Br
replaces CH3
- in replication, 5-bromouracil may become incorporated into DNA in place of thymine, producing an incorporated error.
- 5-Bromouracil may mispair with guanine in the next round of replication.
- in the next replication, this guanine nucleotide pairs with cytosine, leading to a permanent mutation.
- If 5-bromouracil pairs with adenine, no replication error occurs.
CONCLUSION: incorporation of bromouracil followed by mispairing leads to a TA –> CG transition mutation.
Strand separation, replication, Incorporated error, replication, strand separation, replication, replicated error.
Base Ionisation: Understaning what 5-BU ionisation is = 4
- 5-BU ionisation thus causes mispairings of altered
“T” to G … - So 5-BU causes GC (G⋅C) to AT or AT to GC
transitions in the course of replication.
(Recall transitions are purine to purine, or pyrimidine to pyrimidine replacements.) - Which occurs depends on whether the 5-BU was ionised within the DNA, or as an incoming base
- 5-BU’s action as a mutagen is caused by it being in the ionised form.
5-BU transitions - how it works…
GC in DNA, during replication 5-BU(ionised) mispairs with G… now have G5-BU. During replication if 5-BU reverts to common form, then have A5-BU pair.
In next round will have AT … so have GC to AT transition!
Similarly if have A5-BU in the DNA, and 5-BU is ionised, will get G5-BU* after replication, and after next
round GC in DNA … so have an AT to GC transition!
What is 2-AP mutagen?
- 2-AP is a Commonly Used Mutagen
- 2-AP (2-amino purine) is an adenine analogue, which pairs with T.
- Protonated 2-AP pairs with C
2- AP ->T
Protonated 2-AP -> C
Why/ how is 2-AP a commonly used mutagen?
If 2-AP is incorporated into the DNA by pairing with T, it can cause AT to GC transitions, by mispairing with C in subsequent replications.
If 2-AP is incorporated into the DNA by pairing with C, it can cause GC to AT transitions when 2-AP pairs with T in subsequent replications
SO SPECIFIC FOR TRANSITION MUTATIONS
BASE ALTERATION: EMS, NG…
Some mutagens act by altering a base & cause
specific mispairing, eg alkylating agents such as:
ethylmethylsulfonate (EMS) - adds ethyl
& nitrosoguanidine (NG)
- adds methyl.
If alkylation
occurs at oxygen in position 6 of guanine, get O6-
alkylguanine
(fixed so nasty mutagen), which mispairs with T.
Mispairing Causes Mutation: drawing
Should be able to draw this eg tautormeric or wobble base mispairing after two
rounds of rep, where incorporation error and where fixed replication error
pg 11
base ionisation
Be able to draw this! practice!! page 15
What mutation will be present after DNA
replication?
GC pair
EMS
Replication
?
?
?
EMS causes GC to AT transitions
Need to be able to draw after rounds of replication and say it causes transition mutations** page 21
Explain Hydroxylation by Hydroxylamine
Hydroxylamine is a very specific base modifying
mutagen
- adds OH group to cytosine, giving hydroxylaminocytosine, which pairs with A, so will get
CG to TA transitions
Explain Intercalating Agents Modify DNA
Can cause single nucleotide
insertions or deletions –frequently produce frameshift mutations
Egs proflavin, acridine orange, ethidium bromide are all flat, planar molecules that mimic base pairs & slip in between stacked bases (intercalate).
Distort 3D structure of the
double helix
Understanding Base Damage:
- Some mutagens damage one or more bases such that no specific base pairing can occur.
- This results in replication block … bacteria have developed ways to bypass the block by inserting nonspecific bases…resulting in numerous mistakes and high rate of mutation
- In E. coli this requires activation of the SOS REPAIR SYSTEM … this “repair” mechanism can actually mutate the DNA!, In
mammals cells go into APOPTOSIS as we don’t want to carry mutations in our DNA. - SOS is an emergency response to prevent cell death, when DNA is significantly damaged… so it is a mechanism of last resort for the BACTERIAL CELL.
UV light induces RecA synthesis
Error Prone Replication During Translesion Synthesis (red is clamp protein) PolV is error prone polymerase (Translesion DNA synthesis polymerase) highly
expressed in bacterial cells.
RecA binds SSDNA
forming long helical filaments, and causing inactivation of lexA which is a repressor of the SOS system…PolV only expressed during SOS response
(disruptive enzyme – poor replication fidelity hence highly mutagenic)
BASE DAMAGE WITH UV LIGHT
- UV light usually causes mutations, but discovered some E. coli where UV exposure did not cause mutations.
- These mutants
suggested that some E. coli genes actually function to generate mutations on exposure to UV light, and that these were not operating in the mutants. - UV-induced mutation does not occur if have mutated DinB, UmuC or UmuD’ genes. THESE GENES ENCODE ERROR-PRONE DNA POLYMERASES.
Such EP polymerases (sloppy copiers) add nucleotides to the strand opposite the damaged base.
In humans have similar EP polymerases in damage-tolerant mechanism of translesion DNA synthesis (iota and neta).
pol n and pol L HOW THWY WORK?
- Error-prone DNA polymerases bind stalled replication machine.
- binding promoter conformational change. Replication begins. pol n adds erroneous bases.
- Poln dissassociates. Pol L continues incorporating erroneous base on strand opposite damaged base.
- EP polymerases (pol i & pol
h) are always present in
eukaryotes, - Pol h bypasses pyrimidine dimer by inserting AA opposite (2/3 of pyrimidine dimers are thymine dimers).
Insertion of AA opposite CT
dimer results in transversion, better for the cell to have a few mutations than to die!! - PCNA (clamp protein in
humans) ubiquitination
determines affinity of EP pol
…
WHY IS UV A CARCINOGEN? BASE DAMAGE
Mutagens that create bases unable to form stable
pairs rely on SOS-type systems for mutagenic action, as it is the EP polymerases that insert the incorrect nucleotides during replication.
SOS-dependent mutagens include most carcinogens
(e.g. UV light & aflatoxin B1).
UV light induces photoproducts, the most likely to cause mutations are those that unite adjacent pyrimidines on the same strand
Summarise uv light and Base DAMAGE
Disrupts local DNA structure & induce SOS.
Can get C to T
transitions (common), also
transversions, frameshifts &
indels.
UV is broad spectrum type
of mutagen (not only transition)
Fortunately there are REPAIR MECHANISMS!!
Ultraviolet Light Base Damage – Repair mechanisms
Pyrimidine Dimer or 6-4 Photoproduct
–>
No Base Pairing During Replication
–>
REPAIR
OR
SOS Response:
Error Prone Replication
–>
MUTATION
Aflatoxin B1 …Explain how it works
(chemical from fungi – first isolated from fungi on peanuts)
- attaches to N-7 guanine &
- breaks the bond between base & sugar,
= get an apurinic site. - “SOS” (EP pol) often leads to A INSERTION…
- Depurination
- SOS Response:
- Error Prone Replication
- MUTATION
OR - DEPURINATION
- REPAIR
Alfatoxin B1 Metabolism in liver… EXPLAIN
Aflatoxin B1 potent
carcinogen in foods,
- increases the
risk for development of
Hepatocellular carcinoma
(Liver cancer)
Depurination
(E.g. Aflatoxin B1) STEPS 5
- In replication, thye apurinic site cannot provide a tamplate for a complementary base on the newly synthesised strand.
- A nucleotide with the incorrect base (most often A) is incorporated into the newly synthesised strand.
- At the next round of replication, this incorrectly incorporated base will be used as a template…
- …leading to a permanent mutation.
- a nucleotide is incorporated into the newly synthesized strand opposite the apurinic site.
GC TO TA TRANSVERSIONS
