Mar 23, 2016 (Wednesday)

March23,2016

1:11PM

Mutation - Review of Terminology

Many classification schemes
1. Location:
intragenic vs. extragenic
within open reading frames (silent, missense, nonsense, frameshift) <= be able to
distinguish between these four for the exam
Silent: DNA sequence is changed, but amino acid sequence is unchanged (still
codes for the same amino acid) - protein will fold exactly the same way (don't
know that change has happened)
Missense: DNA sequence is changed and amino acid sequence is also changed
(changed from one amino acid to another) - can have significant effect
depending on where amino acid is in the polypeptide sequence
Nonsense: DNA sequence is changed and amino acid is turned into a stop
codon; premature termination of polypeptide
Frameshift: note, if a frameshift leads to a nonsense mutation, it is still only
considered a frameshift, not a nonsense mutation
2. Phenotypic behavior
dominant, recessive, semi-dominant
gain-of function, loss-of function
3. Type of alteration of the DNA sequence
insertion, deletion, point mutations, substitution, rearrangement
double strand breaks, single strand breaks
4. Creation of mutation
spontaneous vs. induced mutations
Definitions
Mutation
a permanent, transmissible change in the DNA (usually a single gene).
Mutagen
a substance that causes an increase in the rate at which mutations occur
Carcinogen
a substance that causes cancer
most carcinogens (> 90%) are mutagens
Transition vs. Transversion Mutation
Use the following terms below when referring to DNA (not point mutation)
Transition
Exchange of a purine to a purine, or pyrimidine to a pyrimindine base. More common than
transversion. Often the result of tautomeric shifts.
GC AT transition
causal agents: e.g. base analog 5bromouracil
AT GC transition
causal agents: e.g. base analog 2-aminopurine
Transversion
Exchange of a purine to a pyrimidine base and vice versa
GC TA or GC CG transversion
AT CG or AT
TA transversion
DNA Damage
1. Polymerase errors

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 Limited by proofreading but errors in pairing and additions/deletions do occur.

Can be due to incorrect dNTPs such as dUTP and 8-oxo-dGTP being incorporated
2. Chemical reactions
Hydrolysis (mainly purines) and deamination (dC, dG, dA)
Oxidation
Alkylation by electrophilic reactants from exogenous/endogenous chemicals
3. Radiation
UV
X-rays

Cause of mutation

Consequence

Remedy

1. Random mismatch

Mutation

Mismatch repair

2. Incorporate dUTP

Not a base in DNA

Base Excision Repair

(BER)

3. Incorporate 8-oxoGTP

Can bind to C or A

BER

(see in-class diagram)

4. Hydrolysis
(depurination)

Breaks glycosidic bond (creates AP BER

site)
Pentose ring opens, creates reactive
aldehyde

5. Base deamination

(see in-class diagram)

BER

6. O6-methylguanine

(see in-class diagram)

BER
Direct repair

7. Intercalation (BaP)

Leads to insertion/deletion

Nucleotide-excision repair
(NER)

8. UV

Stall replication (effects pyrimidine NER

dimer)
Direct repair

(nitroserine?)

Polymerase Errors
Mispairs
Incorporation of dUTP, 8-oxo-dGTP
Insertion/deletions

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Polymerase Error: 8-oxoG Base Pairs

Chemical: Depurination
The N-glycosyl bond between the base and the pentose can undergo hydrolysis.

Chemical: Base Deamination

Nucleotide bases can undergo spontaneous loss of their exocyclic amino groups
(=deamination).

Chemical: Potential Sites of Alkylation Damage

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Chemical: For Example: O6-methylguanine

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Chemical: Bulky adducts, Benzo[a]pyrene (BaP)

Inert BaP is converted to the reactive epoxide in an attempt by the body to make it
hydrophilic so it can be excreted.
The epoxide is an electrophile that will react with nucleophiles on DNA

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Radiation: Adducts from Ultraviolet Radiation

DNA photoproducts formed between adjacent pyrimidines
Base substitutions are at TT, TC, CT, CC sequences

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DNA Repair
Several ways DNA can become damaged:
Incorporation of wrong bases
Damage to existing double-helical DNA
Organisms need to repair their genomic DNA
Several systems exist in all species.
The pattern of repair depends upon the type of damage.
Mismatch repair system
E. coli contains a methylation system that methylates adenine bases in GATC sequences (note:
this is palindromic).
Both strands will be methylated at least once every ~1 kbp.
Before replication, both strands will be methylated.
Immediately after replication, the new strand will not be methylated, as the methylation
system has not had time to act. (DNA will be hemimethylated can distinguish old
strand from new strand for a few minutes before new strand is methylated)
During this delay, the mismatch repair system has the opportunity to recognize
mismatches and target the nonmethylated (new) strand for excision.

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 MutL-MutS will bind to the mismatch

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 MutH binds to MutL-MutS and scans left/right for nearest methylated site
Has endonuclease activity will destroy the new strand

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 MutL-MulS complex is still at mismatch site (not shown)

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 Exonuclease will start eating up until it gets to MutL-MutS complex

Two arms of pathway: simply means that one is eating from right left, and other is left
right (don't need to know specific names, just the specific activities)
This system assumes that the template is correct and the mistake occurred in the
replication step
Also not shown, you need a ligase to seal everything up
Base-excision repair system
Bases that have been damaged or modified are removed by DNA glycosylases.
They catalyze the hydrolysis of the glycosidic linkage between the deoxyribose moiety
and the nucleobase.
A common example is uracil glycosylase, which removes the deamination product of
cytosine.
Each glycosylase is rather specific for the type of base it removes.

The abasic or AP (apurinic/apyrimidinic) site is recognized by the enzyme AP

endonuclease, which will cleave the DNA at a point near the lesion.
DNA Pol I can catalyze repair synthesis (or nick translation) though the AP site.
DNA ligase seals the nick.

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 Broken phosphoester bond is called a "nick" (PolI really likes to bind to nicks)

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 Ligase will seal up the nick once Pol I is done

No one knows how long Pol I will stay on for, it will fall off once it's done
Nucleotide-excision repair system
Many types of lesions cause distortions in the double helical structure. Most common are
pyrimidine dimers, (caused by UV irradiation).
How does this system work?
A specialized DNA excinuclease recognizes the distortion and cuts the DNA strand before
and after the lesion.
DNA helicase removes the fragment containing the lesion.
The gap is repaired by DNA polymerase
DNA ligase seals the nick.

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 The left = prokaryotes (don't need to know specific numbers)

The right arm = eukaryotes
This is the only exception where DNA Pol I does not bind to a nick (binds to small gap in
this case)
Direct Repair
There are some systems devoted to repairing certain common lesions in DNA without
removing bases or nucleotides.
The bases are converted back to their original form while still part of the double helix.
Photolyase

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 Mammals are the only organism that cannot use photolyase to repair DNA
Direct Repair
O6-methylguanine-DNA methyltransferase
Transfers the methyl group from O6- methylguanine to a Cys on the protein.
This inactivates the protein!

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O6-methylguanine-DNA methyltransferase

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