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Mutations & DNA Repair

I. What are Mutations?II. Mutagenesis: Process of producing

a mutationIII. Testing MutagenityIV. Repair of Mutations

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Mutation(G47R)inthexpdgenewithintheNterminusoftheprotein-Xerodermapigmentosum:LackexcisionrepairmechanismandcellssufferfromUVlightdamage,resultinginphotosensitivity,photodamage,andpigmentation,theearlydevelopmentofcutaneousmalignancies

http://www.nature.com/jid/journal/v125/n1/full/5603240a.htmlhttp://www.sciencedaily.com/releases/2008/05/080529120718.htm

I. What are mutations?A. Classes of mutations:

Spontaneous mutation - occurs in nature without the addition of amutagen

Induced mutation – Point mutation –

Transition = pyrimidine for pyrimidine, or purine for purine Transversion = pyrimidine for purine, or purine for pyrimidine

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 (null mutation) Gain of function mutation Adaptive mutation - provides a selective advantage to the organism Deleterious - harmful to the organism

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Base substitutions/Point mutations

• Transitions– Purine replaced by a purine…

or pyrimidine replaced by apyrimidine

• Transversions– Purimidine replaced by a

purine, or vise versa– Less common… why?

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 –

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B. Mutational outcomes

1) Silent substitution – function of theprotein product of gene isunaltered

2) Missense mutation – alters codonso that it encodes a differentamino acid

3) Nonsense mutation – alters geneso that it creates a nonsensecodon (no normal tRNA exists)causing termination of translation

Geneticists Use Mutations to IdentifyGenes and Study Gene Function

• Mutation is usually a non-adaptiveprocess -

• (table 16.2)

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WaysMutationscanoccur:

-Damagetonucleicacids-Mobileelements

II.Mutagenesis:Processofproducingamutation

A.Spontaneous mutations• Spontaneous mutations arise from replication errors &

base modifications, due to natural/biological chemicalprocesses.

1) DNA Replication errors– Replication slippage – one strand loops out and becomes

displaced during replication– DNA pol stuttering– Occurs frequently in repeat regions: each of the bases can appear in one of several forms called

tautomers (isomers)2) Tautomeric Shifts - Tautomerization – isomerization of a

nitrogen base to an

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Tautomerization–Knownasatautomericshift“rare”formsresultinmispairing,.

Spontaneous Mutations continued:

3) Spontaneous lesions– Depurination -– Deamination = ie deamination of C yields U, which

will pair w/A leading to a– Oxidative damage – superoxide radicals (byproducts

of metabolism) alter bases to cause mispairing… 8-oxidG or GO pairs with A

4) Transposable elements– significant part of the genome consists of “nomadic”

DNA sequences that are present at differentlocations

Spontaneous mutation rate various among organisms (table 16.1)

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B. Induced MutationMechanisms

1) Base replacement2) Base alteration3) Base damage

1. Base replacement• Base analogs (chemicals that are similar to

nucleotides) substitute themselves for thenucleotide

• Result =• Examples:

5-Bromocuracil (T analog),2-Aminopurine (A analog)

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Mispairingresultsinreplicationerrors–

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5BU(derivativeofuracil)behavesasathymineanalog,if5BUisincorporated

2. Base alterationChemicals cause the shape

– 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 pairsand slip themselves between the stacked nitrogenbases at the core of the helix• Ethidium bromide• Proflavin• Acridine orange

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EMSalkylatestheketogroupsofGandT

Intercalatingagentsslipbetweenthenitrogenousbases,whichcanleadtoinsertion/deletions.

–generatedatgapsproducedinDNAduringreplication

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3. Base damageChemicals, oxidation, radiation cause the

nucleotide to become modifiedUV light

• Results in

Radiation• Causes ionization of molecules• Creates substitutions• Breaks

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III. Testing mutagenicityA. The Ames Test

Test to determine if a chemicalis a mutagen

suspension of a histidine-requiring (His－) strain ofSalmonella typhimurium platedwith a mixture of rat liverenzymes on agar lackinghistidine

mutagenic effect (reversemutation) of the chemical cancause bacteria to regain theability to grow without histidine,forming the colonies seenaround the disk

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B. Using transgenic mice to testpossible mutagens

• Big Blue mice are transgenic for a segment of DNA thatcontains:• Lambda phage DNA used as a vector

• 3 genetic elements from the lac operon of E. coli• the lacI gene• the operator of the operon• the beta-galactosidase (lacZ) gene

• The transgenic mice are given repeated doses of thesuspected carcinogen for a week or two. If the chemical ismutagenic, it will cause random mutations throughout thegenome of each mouse cell. If a mutation occurs in either

• the lacI gene (which encodes the lac repressor) or theoperator, the gene

• Mouse DNA then extracted and assayed

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IV. Repair of mutations

1. Direct Reversal of damage2. Excision repair3. Proofreading4. Mismatch repair5. Post-replication repair & SOS6. Double-strand break repair

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1. Direct reversal of damagePhotoreactivation repair: reversal of UV damage

Photolyase splits

Photolyase• The two bind together in dark to T-dimer• When light shines on cell

O6-mGua DNA methyltransferase

Alkyltransferase – one time repair enzyme thatremoves ethyl or methyl groups from guanine

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2. Excision repair

Involved in repair of deamination anddepurination

Enzymes recognize an abnormal base andcleave the bond between in and the sugar inthe DNA backbone.

1) Uracil N-glycosylase removes uracil

2) AP endonuclease cuts 5’ side of damaged site on apurinic bases

3) Phosphodiesterase Removes sugar-phosphate residue

AGTGACTTAGTCAUTGAATC

AGTGACTTAGTCA TGAATC

AGTGACTTAGTCA TGAATC

AGTGACTTAGTCACTGAATC

deamination

U

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•BER involves:recognition of theerroneous base byDNA glycosylase•cutting of the DNAbackbone by APendonuclease

NER: repairs bulkylesions and involvesthe uvr genes

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3. Proofreading

DNA Pol III error rate:• Proofreading ability: Pol II can

recognize

• 3’ to 5’ exonuclease ability,

4. Mismatch repair

• Mismatch repair – after proofreading,mismatches identified, improper baseexcised and replaced w/correct base– Adenine methylase recognizes parent

strand and adds methyl group to A’s

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Mismatch repair-

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

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SOS response• Severe damage due to alkylating agents

or cross-linking agents (UV radiation beststudied) triggers this response– Translesional polymerases (POL II, IV, V)

can replicate over damaged regions

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6. Double strand break repair

• Repairs DSBs by reannealing the two DNAsegments - protein aligns the broken ends of DNAfor rejoining

• Recombination repair mechanism– Homologous recombination repair –

– Nonhomologous recombination repair – uses non-homologous region for replacement

• Errors in direct joining may be a cause of translocations