Transcript BIOL 112 – Principles of Zoology

Mutations & DNA Repair
I.
II.
What are mutations?
Mutagenesis: Process of producing a
mutation
III. Repair of mutations
Mutations can cause changes in the shape of a protein
which alters its function.
I.
What are mutations?
A. Classes of mutations:
 Spontaneous mutation - occurs in nature without the
addition of a mutagen
 Induced mutation – caused by a mutagen
 Point mutation – change of 1 nucleotide
 Insertion/Deletion – base added or deleted
 Frameshift mutation – loss or addition of a nucleotide alters
the codon reading frame
 Forward mutation – converts wild type to mutant
 Reverse mutation – converts mutant back to wild type
 Loss of function mutation
 Gain of function mutation
Base substitutions/Point mutations
Transitions
 Purine replaced by a purine…
or pyrimidine replaced by a
pyrimidine
Transversions
 Purimidine replaced by a
purine, or vise versa
 Less common… why?
T&C
A & G
Additional mutational categories:
Lethal mutation – results in death
Conditional mutation – expression depends on the
environment

i.e. temperature sensitive mutations
Somatic mutation – not transmitted to future generations
Germinal/gametic mutation – transmitted to offspring
Mutational outcomes
1)
2)
3)
Silent substitution – function of the
protein product of gene is
unaltered
Missense mutation – alters codon
so that it encodes a different
amino acid
Nonsense mutation – alters gene
so that it creates a nonsense
codon (no normal tRNA exists)
causing termination of translation
Spontaneous mutations
Spontaneous mutations arise from
replication errors & base modifications
DNA Replication errors



Replication slippage – one strand loops out and
becomes displaced during replication
DNA pol stuttering
Occurs frequently in repeat regions: Hot Spots
for DNA mutation
Spontaneous mutation rate various among
organisms (table 15.2)
II. Mutagenesis:
Process of producing a
mutation
Ways Mutations can occur:
-Replication Error
-Breaks in DNA strands
-Damage to nucleic acids
-Mobile elements
Induced Mutation Mechanisms
1) Base replacement
2) Base alteration
3) Base damage
1. Base replacement
Base analogs (chemicals that are similar to
nucleotides) substitute themselves for the nucleotide
Result = improper base pairing
Examples:
a) Tautomeric Shifts - Tautomerization – isomerization
of a nitrogen base to an alternative H-bonding
condition
b) Chemicals:
5-Bromocuracil (T analog),
2-Aminopurine (A analog)
Tautomerization – Known as a tautomeric shift
“rare” forms result in mispairing,
.
Mispairing results in replication errors – the wrong
bases are incorporated into the daughter strands
5 BU (derivative of uracil) behaves as a thymine analog,
if 5 BU is incorporated it will base pair with guanine, after
1 round of replication an A-T to G-C transition results
2. Base alteration
Chemicals cause the shape of the nucleotide to
change, resulting in improper base pairing

Depurination (loss of nitrogenous base) & Deamination
(amino group converted to keto group)
Alkylation – addition of alkyl group (CH3 or CH3CH2)
to bases
•
EMS (ethylmethane sulfonate)
Intercalation – planer molecules that mimic base pairs
and slip themselves between the stacked nitrogen
bases at the core of the helix
•
•
•
Ethidium bromide
Proflavin
Acridine orange
EMS alkylates the keto groups of G and T, base pairing
is altered and a transition results
Intercalating agents slip between the nitrogenous bases,
which can lead to insertion/deletions.
Frameshift mutations result – generated at gaps
produced in DNA during replication
3. Base damage
Chemicals, oxidation, radiation cause the
nucleotide to become modified in such a
way that it can no longer base pair
UV light
•
Results in pyrimidine dimers
Radiation
•
•
•
Causes ionization of molecules
Creates substitutions
Breaks phosphodiester bonds
Spontaneous Mutations– arise due to natrual
biological/chemical processes:
DNA replication errors

DNA replication errors = each of the bases can
appear in one of several forms called tautomers
(isomers)
Spontaneous lesions



Depurination - Apurinic sites can’t specify a base
complementary to the original
Deamination = ie deamination of C yields U, which
will pair w/A leading to a GC to AT transition
Oxidative damage – superoxide radicals (byproducts
of metabolism) alter bases to cause mispairing… 8oxidG or GO pairs with A
Transposable elements

significant part of the genome consists of “nomadic”
DNA sequences that are present at different locations
III. Repair of mutations
1.
2.
3.
4.
5.
6.
Direct Reversal of damage
Excision repair
Proofreading
Mismatch repair
Post-replication repair & SOS
Double-strand break repair
1. Direct reversal of damage
Photoreactivation repair: reversal of UV damage
Photolyase splits Thymine dimers, restoring DNA to
its original condition

Photolyase works with cofactor folic acid
 The two bind together in dark to T-dimer
 When light shines on cell –folic acid absorbs the light &
uses the energy to break the covalent bond between T’s

O6-mGua DNA methyltransferase
Alkyltransferase – one time repair enzyme that
removes ethyl or methyl groups from guanine
2. Excision repair
Involved in repair of deamination and
depurination
Enzymes recognize an abnormal base and
cleave the bond between in and the sugar in
the DNA backbone.
1)
Uracil N-glycosylase

2)
AP endonuclease

3)
removes uracil
cuts 5’ side of damaged site on apurinic bases
Phosphodiesterase

Removes sugar-phosphate residue
deamination
AGTGACTTAG
TCACTGAATC
AGTGACTTAG
TCAUTGAATC
Ligase
Uracil N-clycosylase
U
AGTGACTTAG
TCA TGAATC
AGTGACTTAG
TCA TGAATC
Pol I
3. Proofreading
DNA Pol III error rate: 10-5
Proofreading ability: Pol II can
recognize mismatched base pairs,
determine which base is the incorrect
one excise the wrong base and carry
out repair synthesis
3’ to 5’ exonuclease ability, lowering the
error rate to 10-7
4. Mismatch repair
Mismatch repair – after proofreading,
mismatches identified, improper base
excised and replaced w/correct base


Adenine methylase recognizes parent strand
and adds methyl group to A’s
Unmethylated daughter strand recognized by
repair enzyme
Mismatch repairImportant to recognize
difference between old
strand & new strand:
If mutated base excised,
the wild type is restored,
but if the original wild type
is excised, the mutant
sequence becomes fixed.
5. Post-replicational repair & SOS
Post-replicational repair (aka recombination
repair ):
Damaged DNA cause Pol III to “stutter” and
skip past damaged site
Replication restarts downstream and a gap is
left
Gap is repaired by retrieving sequence from
the normal copy and then the subsequent gap
is repaired
SOS response
Severe damage due to alkylating agents
or cross-linking agents (UV radiation best
studied) triggers this response


Translesional polymerases (POL II, IV, V)
can replicate over damaged regions
Has very high error rate: 10-2, however
allows for survival under extremely bad
conditions
6. Double strand break repair
Repairs DSBs by reannealing the two DNA
segments - protein aligns the broken ends
of DNA for rejoining
Recombination repair mechanism


Homologous recombination repair – damaged
DNA replaced by homologous DNA section from
sister chromatid
Nonhomologous recombination repair – uses
non-homologous region for replacement
Errors in direct joining may be a cause of
translocations