Mutation Repair

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Mutation - Review of Terminology

intragenic vs. extragenic○

Silent: DNA sequence is changed, but amino acid sequence is unchanged (stillcodes for the same amino acid) - protein will fold exactly the same way (don'tknow 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 effectdepending on where amino acid is in the polypeptide sequence

Nonsense: DNA sequence is changed and amino acid is turned into a stopcodon; premature termination of polypeptide

Frameshift: note, if a frameshift leads to a nonsense mutation, it is still onlyconsidered a frameshift, not a nonsense mutation

within open reading frames (silent, missense, nonsense, frameshift) 90%) are mutagens•

Carcinogen

Use the following terms below when referring to DNA (not point mutation)•Transition vs. Transversion Mutation

Transition

causal agents: e.g. base analog 5’bromouracil○GC AT transition

causal agents: e.g. base analog 2-aminopurine○AT GC transition

Exchange of a purine to a purine, or pyrimidine to a pyrimindine base. More common thantransversion. Often the result of tautomeric shifts.

Transversion

GC TA or GC CG transversionAT CG or AT TA transversion

Exchange of a purine to a pyrimidine base and vice versa

DNA Damage

1. Polymerase errors

Mar 23, 2016 (Wednesday)March 23, 2016 1:11 PM

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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•

Hydrolysis (mainly purines) and deamination (dC, dG, dA)•Oxidation•Alkylation by electrophilic reactants from exogenous/endogenous chemicals•

2. Chemical reactions

UV•X-rays•

3. Radiation

Cause of mutation Consequence Remedy

Random mismatch1. Mutation Mismatch repair•

Incorporate dUTP2. Not a base in DNA Base Excision Repair(BER)

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Incorporate 8-oxoGTP3. Can bind to C or A

(see in-class diagram)

BER•

Hydrolysis(depurination)

4. Breaks glycosidic bond (creates APsite)

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Pentose ring opens, creates reactivealdehyde

•

BER•

Base deamination5. (see in-class diagram) BER•

O6-methylguanine(nitroserine?)

6. (see in-class diagram) BER•Direct repair•

Intercalation (BaP)7. Leads to insertion/deletion• Nucleotide-excision repair(NER)

•

UV8. Stall replication (effects pyrimidine

dimer)

• NER•

Direct repair•

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

Polymerase Errors

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Polymerase Error: 8-oxoG Base Pairs

Chemical: DepurinationThe 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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Inert BaP is converted to the reactive epoxide in an attempt by the body to make ithydrophilic so it can be excreted.

•

The epoxide is an electrophile that will react with nucleophiles on DNA•

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

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

Radiation: Adducts from Ultraviolet Radiation

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DNA Repair

Incorporation of wrong bases•Damage to existing double-helical DNA•

Several ways DNA can become damaged:

Several systems exist in all species.•The pattern of repair depends upon the type of damage.•

Organisms need to repair their genomic DNA

Mismatch repair system

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 methylationsystem has not had time to act. (DNA will be hemimethylated→ can distinguish oldstrand from new strand for a few minutes before new strand is methylated)

•

During this delay, the mismatch repair system has the opportunity to recognizemismatches and target the nonmethylated (new) strand for excision.

•

E. coli contains a methylation system that methylates adenine bases in GATC sequences (note:this is palindromic).

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MutL-MutS will bind to the mismatch•

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Has endonuclease activity→will destroy the new strand○

MutH binds to MutL-MutS and scans left/right for nearest methylated site•

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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)

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This system assumes that the template is correct and the mistake occurred in thereplication step

•

Also not shown, you need a ligase to seal everything up•

Bases that have been damaged or modified are removed by DNA glycosylases.•They catalyze the hydrolysis of the glycosidic linkage between the deoxyribose moietyand the nucleobase.

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A common example is uracil glycosylase, which removes the deamination product ofcytosine.

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Each glycosylase is rather specific for the type of base it removes.•

Base-excision repair system

The abasic or AP (apurinic/apyrimidinic) site is recognized by the enzyme APendonuclease, which will cleave the DNA at a point near the lesion.

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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•

Many types of lesions cause distortions in the double helical structure. Most common arepyrimidine dimers, (caused by UV irradiation).

•

Nucleotide-excision repair system

A specialized DNA excinuclease recognizes the distortion and cuts the DNA strand beforeand after the lesion.

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DNA helicase removes the fragment containing the lesion.•The gap is repaired by DNA polymerase•DNA ligase seals the nick.•

How does this system work?

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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 inthis case)

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There are some systems devoted to repairing certain common lesions in DNA withoutremoving bases or nucleotides.

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The bases are converted back to their original form while still part of the double helix.•

Direct Repair

Photolyase

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Mammals are the only organism that cannot use photolyase to repair DNA•

Transfers the methyl group from O6- methylguanine to a Cys on the protein.•

This inactivates the protein!•

Direct Repair

O6-methylguanine-DNA methyltransferase

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O6-methylguanine-DNA methyltransferase

Documents

Mutation Repair