MBB 267 Week 5: Corrigan 2

Question 1

What is a mutagen?

Answer 1

Mutation causers

Question 2

What are examples of mutagens?

Answer 2

Include:

a. Electromagnetic radiation
b. Spontaneous tautomers during replication (Structural ismoers of compounds/molecules)
c. Chemicals;
i. Analogs of bases = look-alike molecules
ii. Base-modifying chemicals; Nitrosoguanidine, nitrous acid
iii. Intercalators insert between base; Cause frameshift mutations

Question 3

Different types of mutations?

Answer 3

Include;

a. Silent
b. Missense mutation
i. Change one codon to another
c. Nonsense mutations
i. Change a codon to Stop
d. Frameshift mutation
i. Insert or delete a single base
ii. Changes bases read by ribosome
iii. Alters all codons downstream of mutation

Question 4

What is slip strand mispairing?

Answer 4

a mutation process which occurs during DNA replication
a. When there is a repetitive sequence and the sequence aligns incorrectly. The slipped DNA will have another repeat.

Question 5

What is the use of slip strand mis pairing?

Answer 5

Used in some pathogenic bacteria to switch expression of surface exposed proteins on or off for immune evasion – PHASE VARIATION

Question 6

How can insertion/deletion of DNA happen?

Answer 6

3 types

a. Recombination
i. Homologous; alleles swapped
ii. Illegitimate/non-homologous; strands are not lined properly together
b. Site-specific
i. Use lambda-integrase to insert DNA fragment in another DNA genome
c. Transposable elements use replicative recombination

Question 7

Error-proof DNA repair mechanisms?

Answer 7

Include:
a. Methyl mismatch repair
– Mis-paired base cut out of strand
b. Nucleotide excision repair – thymine dimers – Induced by UV
c. Base excision repair - damaged bases – Excised by specific enzymes
d. Recombinational repair

Question 8

How does methyl mismatch repair work?

Answer 8

A mismatch in the double stranded DNA isn’t connected

a. Enzyme MutS binds to DNA mismatch
b. MutS attracts MutHL to the mismatch site
c. MutL binds to an adjacent hemimethylated GATC site
d. Causes looping of DNA
e. MutH cleaves unmethylated strand at the 5’ of the GATC
f. UvrD unwids the nicked strand
g. Exonuclease removes damaged strand
h. Polymerase I synthesis a new strand

Question 9

What is a thymine dimer?

Answer 9

A pair of abnormally chemically bonded adjacent thymine bases in DNA, resulting from damage by ultra-violet irradiation.

Question 10

How are thymine dimers repaired?

Answer 10

Thymine dimer in DNA

a. UvrAB form a complex and bind to damaged DNA
b. UvrA bends DNA then it leaves
c. UvrB attracts UvrC to damaged site
d. UvrC cleaves the phosphodiester backbone of the damaged backbone at 2 places.
e. UvrD helicase strips the cut region
f. DNA pol I fills the gap
g. DNA ligase seals

Question 11

How does base excision repair work?

Answer 11

Damaged base

a. DNA glycolase binds to damaged site
b. Excises damaged base
c. Endonuclease cleaves phophodiester backbone
d. DNA pol synthesises new strand
e. DNA ligase seals DNA

Question 12

How does recombinational repair work?

Answer 12

Part of the DNA is missing

a. How it can happen;
i. Replication fork approaches thymine dimer
ii. DNA polymerase skips damaged region
b. How it is fixed
i. The RecA proteins binds to the sister doubles helices at the missing segment
ii. The undamaged DNA part is cut and placed in the gap (D loop forms)
iii. Gap in undamaged strand is repared by DNA pol
iv. Thymine dimer can be fixed

Question 13

When does error-prone repair happen?

Answer 13

Example; SOS repair

a. Extensive DNA damage inactivates LexA
b. Activation of many repair genes
c. Rapid polyermisation of DNA
i. Last resort repair
ii. Promotes mutations

Question 14

How does error-prone repair work?

Answer 14

The repressor LexA binds to target genes which turns the SOS off

a. DNA damage causes accumulation of ssDNA = SOS system turns on
i. RecA binds ssDNA. Coprotease is activated. LexA is auto-digested.
b. In the absence of LexA, the SOS genes are transcribed and DNA damage is repaired.
i. Products of the target genes inhibit cell division, mediate replication across lesions (Pol V) and participate in nucleotide excision repair.

Question 15

What are the genes included in the SOS system?

Answer 15

Include;

a. SulA = inhibitor of cell division
b. umuDC = Pol V (repair enzyme), error prone
c. uvrA
d. lexA = repressor

Question 16

What is mutation rate?

Answer 16

estimation of the probability of a mutation occurring per cell division

Question 17

How is the mutation rate calculated?

Answer 17

number of mutations divided by the number of cell divisions

Question 18

What is mutation frequency?

Answer 18

ratio of number of mutants total cells

Question 19

How can bacterial mutants be selected using phenotypic selection?

Answer 19

2 types
a. Direct selection procedures - Use media that only allows growth of mutant colonies.
Very powerful: Select for resistance to some agent e.g. antibiotics, toxic analogues for carbohydrates, amino acids, nucleotide metabolism.
i. Wild-type – killed; Mutants in transport or metabolism –form colonies on plates containing the agent.
b. Test every bacterium!
i. e.g. on Indicator media - pH changes; chromogenic substrates Replica plating is used to detect mutants conditional on temperature or medium, e.g. nutritional auxotrophs

Question 20

What are examples of direct selection procedures?

Answer 20

Examples:

a. ONPG (orthonitrophenyl galactoside) is a lactose analogue that is toxic if it is transported or metabolised by the cell (lacY and lacZ mutants).
b. Streptomycin is an antibiotic that binds to the ribosome to inhibit protein translation

Question 21

What is the difference between selection and screening for mutant cells? What are the advantages and disadvantages of each approach?

Answer 21

In direct selection, we use media that only allows the growth of mutants

i. It is easy to find mutants
ii. Quick
b. Screening Is testing every bacterium
i. Very helpful to determining many mutants
ii. Slow