18 - DNA mutation and repair*

Question 1

Gene

Answer 1

DNA sequence encoding for a polypeptide, tRNA or rRNA

Question 2

Genotype*

Answer 2

Collection of genes an organism has (genetic composition)

Question 3

Phenotype*

Answer 3

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

Question 4

Mutation

Answer 4

A permanent, heritable change to the base sequence of DNA

Question 5

Wild type

Answer 5

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

Question 6

Mutant

Answer 6

Organism that differs from wild type as a result of mutation

Question 7

How often do mutations occur

Answer 7

1 in 10^7 to 10^11 base pairs

Question 8

3 types of mutagens

Answer 8

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

Question 9

DNA modifying agents

Answer 9

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

Question 10

Intercalating agents

Answer 10

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

Question 11

UV Thymine dimers

Answer 11

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

Question 12

Oxygen radicals

Answer 12

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

Question 13

Point mutations

Answer 13

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

Question 14

Transition base substitution

Answer 14

Purine replaced with purine or pyrimidine replaced with pyrimidine

Question 15

Transversion base substitution

Answer 15

Purine replaces pyrimidine or pyrimidine replaces purine

Question 16

Greater than one base change mutations

Answer 16

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

Question 17

Effects of DNA mutation on encoded protein

Answer 17

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

Question 18

Silent mutation

Answer 18

No effect on protein (or amino acid sequence)

Question 19

Missense

Answer 19

amino acid replaced by different amino acid

Question 20

Conservative missense

Answer 20

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

Question 21

Non-conservative missense

Answer 21

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

Question 22

Nonsense mutation

Answer 22

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

Question 23

Frameshift

Answer 23

Caused by nucleotide deletion of insertion which changes reading frame

Question 24

Revertant

Answer 24

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

Question 25

True reversion (back mutation)

Answer 25

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

Question 26

Suppression (2nd site mutation)

Answer 26

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

Question 27

Intragenic suppression

Answer 27

second mutation is in same gene as first

Question 28

Extragenic/intergenic suppression

Answer 28

second mutation is in different gene from first

Question 29

Direct detection of mutants

Answer 29

Screening (visual observation)

Question 30

Disadvantages of screening

Answer 30

Labour intensive and not all mutations can be visually detected

Question 31

Indirect detection of mutants

Answer 31

Replica plating and selection

Question 32

Replica painting example

Answer 32

- 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

Question 33

Selection

Answer 33

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

Question 34

other examples of selectable mutants

Answer 34

- bacteriophage resistant mutant - temperature resistant mutant - prototrophic mutant

Question 35

Direct repair

Answer 35

Restoration to original undamaged state

Question 36

Examples of direct repair

Answer 36

- Photoreactivation - Nucleotide excision repair (short patch repair) - Mismatch repair

Question 37

Indirect repair

Answer 37

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

Question 38

Nucleotide excision repair

Answer 38

- 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

Question 39

Mismatch repair at 3' end of replicating DNA

Answer 39

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

Question 40

Mismatch repair not at 3' end

Answer 40

- 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

Question 41

Recombinational repair

Answer 41

- 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

Question 42

Holliday junctions

Answer 42

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

Question 43

Branch migration of holliday junctions

Answer 43

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

Question 44

How do two DNA molecules separate in a holliday junction

Answer 44

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