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Mutations & DNA Repair. What are mutations? Mutagenesis: Process of producing a mutation Repair of mutations. Mutations can cause changes in the shape of a protein which alters its function. What are mutations?. Classes of mutations:

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mutations dna repair
Mutations & DNA Repair
  • What are mutations?
  • Mutagenesis: Process of producing a mutation
  • Repair of mutations
what are mutations
What are mutations?
  • 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
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
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
Mutational outcomes
  • 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
  • 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)
slide8

Ways Mutations can occur:

-Replication Error

-Breaks in DNA strands

-Damage to nucleic acids

-Mobile elements

II. Mutagenesis: Process of producing a mutation

induced mutation mechanisms
Induced Mutation Mechanisms
  • Base replacement
  • Base alteration
  • Base damage
1 base replacement
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)

slide11

Tautomerization – Known as a tautomeric shift

“rare” forms result in mispairing,

.

slide12

Mispairing results in replication errors – the wrong bases are incorporated into the daughter strands

slide13

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
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
slide16

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
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 mutation s arise due to natrual biological chemical processes
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… 8-oxidG 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
III. Repair of mutations
  • Direct Reversal of damage
  • Excision repair
  • Proofreading
  • Mismatch repair
  • Post-replication repair & SOS
  • Double-strand break repair
1 direct reversal of damage
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
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.

  • Uracil N-glycosylase
    • removes uracil
  • AP endonuclease
    • cuts 5’ side of damaged site on apurinic bases
  • Phosphodiesterase
    • Removes sugar-phosphate residue
slide23

deamination

AGTGACTTAG

TCACTGAATC

AGTGACTTAG

TCAUTGAATC

Ligase

Uracil N-clycosylase

U

AGTGACTTAG

TCA TGAATC

AGTGACTTAG

TCA TGAATC

Pol I

3 proofreading
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
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
slide26

Mismatch repair-

Important 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
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
SOS response
  • Severe damage due to alkylating agents or cross-linking agents (UV radiationbest 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
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
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