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DNA REPAIRAND

MUTATION

Mutations and mutants

Mutation: genetically inheritable change inone or more genes

Change in DNA sequence

Often leads to change in function of gene product

• Wild-type (wt): normal (not mutated)

• Mutant: organism that has a change(mutation) in its genome

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Mechanism of Mutation

• Background (or spontaneous)– Chemical instability of bases in DNA

– Errors in DNA replication

• Induced– Physical (ultraviolet light or ionizing radiation)

– Chemical (chemical carcinogens)

Spontaneous Mutation Rate

• Rate differs for different genes– Size dependence– Sequence dependence– Hot spots

• On average 1 in 100,000 chance of acquiring amutation in a gene each round of replication.

• Each individual has multiple new mutations.

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Induced Mutations

• Chemicals and radiation can cause mutations.

• Chemicals causing mutations are called mutagens.

• Chemicals causing cancer are called carcinogens.

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Alkylating agents

Acridine dyes

Xrays

UV radiation

remove a base

add or remove base

break chromosomes

delete few nucleotides

creates thymidine dimers

Point Mutation• A point mutation is a change of a single nucleotide to one of

the other three possible nucleotides

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Transitionpurine replaces purine

A G or G A•• pyrimidine replaces pyrimidine• C T or T C••••

Transversionpurine replaces pyrimidine orpyrimidine replaces purine

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A or GT or C

T or CA or G

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Missense Mutation

• A point mutation that exchanges one codon for anothercausing substitution of an amino acid

• Missense mutations may affect protein functionseverely, mildly or not at all.

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• Hemoglobin mutation

• glutamic acid valine causes sickle cell anemia

Nonsense Mutation

• A point mutation changing a codon for an amino acid intoa stop codon (UAA, UAG or UGA).

• Premature stop codons create truncated proteins.

• Truncated proteins are often nonfunctional.

• Some truncations have dominant effects due to interferencewith normal functions.

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Insertion or Deletion Mutations

• The genetic code is read in triplet nucleotides duringtranslation.

• Addition or subtraction of nucleotides not in multiples ofthree lead to a change in the reading frame used fortranslation. Amino acids after that point are different, aphenomenon called a frameshift.

• Addition or subtraction of nucleotides in multiples of threeleads to addition or subtraction of entire amino acids butnot a change in the reading frame.

Not all mutations impact proteinfunction

• Silent mutations are mutations that do not alter theamino acid encoded.

AAA and AAG both encode the amino acid lysine.

A mutation from AAA to AAG in a gene alters the DNAsequence but protein sequence remains unchanged.

These codons are called synonymous codons.

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Not all mutations impact proteinfunction

• Missense mutations are those that alter the encoded aminoacid to another amino acid.

• The alteration creates a nonsynonymous codon.

Some nonsynonymous mutations are conservative;chemically similar amino acid may not alter function

The impact of a missense mutation is not predictablefrom protein sequence alone.

DNA Repair

• Errors in DNA replication or damage to DNA createmutations.

• Most errors and damage are repaired by the cell.

• The manner in which DNA repair occurs depends upon thetype of damage or error.

• Different organisms vary in their ability to repair DNA.

• In humans, mutations in DNA replication occur in 1in 100 million bases.

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DNA Repair

There are many different sources and types of DNA damageand the cell contains many different systems to survey for andcorrect DNA damage.

• Mismatch repair

• Excision repair

• Inducible repair

Evidence of DNA Repair

No repairDNA damage

Repair

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Deaminating Agents

Hydroxylamine: Removes amino group of cytosine-------GC to AT

Bisulfite: Deaminate only Cytosine--------Site-directed mutagenesis

Nitrous Acid: Deaminates CytosineAdenine, and Guanine

--------GC to ATAT to GC

Base deamination: Potentially MutagenicDNA damage

How to Repair?

Hypoxanthine Cytosine

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Deamination of Adenine to Hypoxanthine leads tomispairing with Cytosine

Failure to repair a deaminated base leads to point mutation

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Failure to repair abasic sites leads to deletions

DNA Glycosilase: break glycosylbond between damaged base andthe sugar in the nucleotide

AP Endonuclease: cut sugar-phosphatebackbone of DNA

Apurinic site-----A or GApyrimidinic site------C or T

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Deaminated basesare repaired by abase excision mechanism.

Spontaneously occuringabasic sites are repairedby the same mechanism

H

H

N

N

H

NH2

H

O

P

H

O

O

O

O-

O

H

H

HN

NO

H

O

H

O

P

O

O

O-

O

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O

H

H

H

O

O

H

O

P O-

O

H

H

dCMPdUMP

H2 O

NH3

UO

H

N glycosylase

H

HH

O

H

O

P

O

O

O-

OH

H

endonucleasecleavage

Deamination

O

Alkylation

Ethyl methanosulfonate (EMS)

Methyl Methanosulfonate (MMS)

N-methyl-N’-nitro-N-nitrosoguanidine(nitrosoguanidine)

Repair:

*N-Glycosylase remove alkylatedbase.

*Apurinic/apyrimidinic DNA strandis cut by AP endonuclease

*Exonuclease degrade the cut strand

*DNA Pol I resynthesized correctDNA strand

Also: by methyltransferase----remove alkyl

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N

N

O

NH 2N

O

H

HH

HHO

PO

O-

HO

O-

CH 3

NH

O 6 M eGuanine nucleotide

NH

N

N

O

NH 2N

O

H

HH

HHO

PO

HO

O-

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A lkylating agents

O 6 alkyltransferase

O 6 alkyltransferase-M e

Direct Repair:

Methyltransferase

O-

Adaptive Response

Ada : methyltransferase----remove methyl or ethyl group

Ada protein regulatory protein of adaptive respons

genes

alkA: N-glycosylase~remove alkilated base

aidB: increases the resistance of the cells to some

methylating agents. To detoxyfy alkylating agents

alkB: increses the resistance of cells to some alkylating

agents.

Regulation of Adaptive Response

A few copies of the Ada protein normally exist in the cell.

DNA damage by alkylation------Ada protein (Methyl

Transferase) transfer alkyl group.

-from damaged DNA phosphates to itself: activator

-from damaged base, inactivating itself.

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Pyrimidine Dimers

UV irradiation-----Pyrimidine dimerDNA Damage

Thymine dimer---5-, 6-Carbon linkto form a cyclobutane ring

6-Thymine-4 Cytosine

DNA Repair: Photoreactivation

UV light

NH

NH

O

O

NH

NH

O

O

Cyclobutane dimer

NH

NH

NH2

O

NH

NH

O

O

OH

6-4 photoproduct

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The major form of damage caused by UV irradiation isThymine dimers:

Photoreactivation

~Enzyme: Photolyase~contains FADH2group. It absorbs light of wavelengthbetween 350~500 nm.

~Bind and then separate the fused bases

~Light absorbsion: Photolyase separatethe fused bases

~Other mechanism:N-Glycosilase; AP EndonucleaseResynthesize DNA: DNA Pol

Photolyase

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Chemical Mutagens

Most chemical mutagens cause a modfications of the bases.Often these are small adducts (eg. methyl groups) but in thecase of some of the more powerful mutagens, these adducts canbe bulky.

Also, many mutagens are polycyclic compounds thatintercalate into the stacked bases of the double helix.

E.g. benzopyrene

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Nucleotide Excision Repair

*Very efficient*Non-specific*Repair many types of damage*Very important: ability the cell tosurvive damage to its DNA

*Repair all types of damage caused byUV irradiation: Cyclobutane dimers;6-4 lesion; base-sugar cross-links.

Mechanism of Repair?

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Mechanism of Repair

Enzyme: UvrABC

*UvrA protein forms a complex withUvrB protein.*The protein complex bind nonspecifically

to the DNA*This complex then migrates up and downthe DNA until the DNA damage

*UvrB binds to the damage, and UvrAleave. UvrA is replaced by UvrC

*UvrC binds UvrB-----UvrB cuts theDNA 4 nucleotides 3’ of the damage

*UvrC cuts the DNA 7 nucleotides 5’ ofthe damage.*UvrD helicase removes oligonucleotidecontaining the damage

*DNA Pol I resynthesizes the strand thatwas removed, using complementarystrand as a template

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Dimeric bases andbulky lesions, eg.large chemical adductsare repaired byNucleotide excision repair

Methyl-Directed Mismatch repair

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Deamination of Cytosine creates a G-U mismatchEasy to tell that U is wrong

Deamination of Cytosine creates a G-T mismatchNot easy to tell which base is the mutation.

About 50% of the time the G is “corrected” to Aresulting in a mutation

Damage Due to Reactive O2

Superoxide radicalsHydrogen peroxideHydroxyl radicals

Normal Cellular ReactionUV irradiation ChemicalsHydroxyl radicals

Induce genes for: Catalase

Superoxide dismutasePeroxide reductase

also: Genes to repair the oxidative to DNAcaused by the reactive form of O2

Mutagenic lesion in DNA causedBy reactive oxygen: 8-oxoG or GO

N-Glycosylase

N-Glycosylase

Phosphatase

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Another example:Aflatoxin

Natural product producedby the fungus Aspergillus.

General Repair Mechanisms

Base Analogs

Transition Mutation

Methyl-directed mismatchrepair system

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Frameshift Mutagen

Acridine dye (9-aminoacridine);Proflavin; Ethidium bromide;Aflatoxins (Produced by fungi)

intercalate between basesIn the same strand of the DNA

Deletion of base pairAdding a base pair