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MICR2208 > 18 - DNA mutation and repair* > Flashcards

18 - DNA mutation and repair* Flashcards

1
Q

Gene

A

DNA sequence encoding for a polypeptide, tRNA or rRNA

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2
Q

Genotype*

A

Collection of genes an organism has (genetic composition)

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3
Q

Phenotype*

A

Observable characteristics of an organism (expression of genes). One phenotype has multiple possible genotypes

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4
Q

Mutation

A

A permanent, heritable change to the base sequence of DNA

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5
Q

Wild type

A

Organism as it was first isolated from nature (normal type)

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6
Q

Mutant

A

Organism that differs from wild type as a result of mutation

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7
Q

How often do mutations occur

A

1 in 10^7 to 10^11 base pairs

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8
Q

3 types of mutagens

A
  • DNA modifying agents
  • Intercalating agents
  • Physical agents (Thymine dimers, oxygen radicals)
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9
Q

DNA modifying agents

A

Add alkyl groups to bases and change base pairing. e.g. ethylmethane sulphonate (EMS) adds ethyl groups

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10
Q

Intercalating agents

A

Planar compounds which insert into DNA helix and distort the backbone (e.g. acridine orange, ethidium bromide)

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11
Q

UV Thymine dimers

A

UV induced Intra-strand pyrimidine dimers (mainly T-T) that distort double helix and prevent replication

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12
Q

Oxygen radicals

A

Cause single and double stranded breaks, prevents replication (e.g. gamma and x rays)

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13
Q

Point mutations

A
  • Base substitution (transversion and transition)
  • Base addition or deletion (indel)
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14
Q

Transition base substitution

A

Purine replaced with purine or pyrimidine replaced with pyrimidine

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15
Q

Transversion base substitution

A

Purine replaces pyrimidine or pyrimidine replaces purine

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16
Q

Greater than one base change mutations

A
  • Addition or deletion of multiple bases
  • Inversion of segment of DNA
  • Duplication of segment of DNA
  • Translocation
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17
Q

Effects of DNA mutation on encoded protein

A
  • Synonymous (silent) mutation
  • Missense mutation (conservative and non-conservative)
  • Nonsense mutation
  • Frameshift mutation
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18
Q

Silent mutation

A

No effect on protein (or amino acid sequence)

19
Q

Missense

A

amino acid replaced by different amino acid

20
Q

Conservative missense

A

Replacement with an amino acid of similar biochemical profile. No loss in protein function

21
Q

Non-conservative missense

A

Replacement with an amino acid of different biochemical profile. Complete or partial loss of function (leaky mutant)

22
Q

Nonsense mutation

A

gives rise to stop codon (UAA, UGA, UAG). Limited effect (if close to end of reading frame) or complete loss of function (truncated protein)

23
Q

Frameshift

A

Caused by nucleotide deletion of insertion which changes reading frame

24
Q

Revertant

A

a strain in which a 2nd mutation has restored the phenotype altered by a 1st mutation

25
Q

True reversion (back mutation)

A

Original sequence is restored. Point mutations revert, large deletions do not.

26
Q

Suppression (2nd site mutation)

A

A change at a different site in the genome that phenotypically corrects the 1st mutation which is still present. (intragenic and intergenic/extragenic)

27
Q

Intragenic suppression

A

second mutation is in same gene as first

28
Q

Extragenic/intergenic suppression

A

second mutation is in different gene from first

29
Q

Direct detection of mutants

A

Screening (visual observation)

30
Q

Disadvantages of screening

A

Labour intensive and not all mutations can be visually detected

31
Q

Indirect detection of mutants

A

Replica plating and selection

32
Q

Replica painting example

A
  • Auxotroph has defect in a biosynthetic pathway (prototroph is wild type)
  • Unable to synthesise an essential nutrient
  • Unable to grow unless supplied with that nutrient
  • Two plates compared, one with and one without nutrient
33
Q

Selection

A

use incubation conditions under which the mutant will grow but the wild-type will not (e.g. antibiotic resistance)

34
Q

other examples of selectable mutants

A
  • bacteriophage resistant mutant
  • temperature resistant mutant
  • prototrophic mutant
35
Q

Direct repair

A

Restoration to original undamaged state

36
Q

Examples of direct repair

A
  • Photoreactivation
  • Nucleotide excision repair (short patch repair)
  • Mismatch repair
37
Q

Indirect repair

A

Damage bypass system using DNA replication (e.g. recombination repair)

38
Q

Nucleotide excision repair

A
  • Products of uvrA, uvrB and uvrC genes form UvrABC endonuclease
  • UvrAB complex migrates up and down DNA
    until it hits a distortion e.g. thymine dimer
  • UvrA released
  • UvrC binds and cuts DNA
  • UvrD helicase removes damaged fragment and releases UvrBC complex
  • DNA polymerase 1 fills in gap and DNA ligase joins fragments
  • ~13nt replaced
39
Q

Mismatch repair at 3’ end of replicating DNA

A

DNA polymerase 3 has 3’-5’ exonuclease activity (proof reading/editing function)

40
Q

Mismatch repair not at 3’ end

A
  • MutS protein slides along DNA until mismatch is found.
  • MutL binds MutS and that complex binds to MutH, which is already bound to hemimethylated sequence
  • MutH makes cut in nonmethylated strand and base mismatch is digested
  • DNA polymerase fills in and ligase seals ends
41
Q

Recombinational repair

A
  • DNA replication stalls at thymine dimer and gap is produced which is lethal to cell
  • RecA protein binds to ss gap and initiates recombination which fills gap by removing the correct homologous DNA from the donor DNA leaving a new gap
  • Gap filled by DNA polymerase copying
    undamaged strand, and DNA ligase
  • Dimer remains but cell survives and can use NER
42
Q

Holliday junctions

A

branched nucleic acid structure that contains four double-stranded arms joined. Occurs during homologous recombination

43
Q

Branch migration of holliday junctions

A

Holliday junctions move up and down the DNA by breakage and rejoining of H bonds between bases. Can happen spontaneously but is sped up by ATP hydrolysing RuvAB proteins

44
Q

How do two DNA molecules separate in a holliday junction

A
  • Linear DNA: branch migrates off the end
  • Circular DNA: RuvC protein cuts them apart

MICR2208 (26 decks)

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