LECTURE 12

Question 1

what are the important regions of a bacterial gene?

Answer 1

promoter
antileader (on template strand)
coding region
antitrailer
terminator

Question 2

in what way is RNA made, and what strand is it identical to?

Answer 2

made in 5’-3’ direction, identical to non template strand

Question 3

what are the different types of mutations in DNA (5)?

Answer 3

base substitution
deletion
inversion
transposition
duplication

Question 4

what can mutations lead to?

Answer 4

morphological
- colonial or cellular
lethal mutations (essential) genes
biochemical mutations (pathways)
resistant mutant (antibiotic, chemicals)

Question 5

how can the cell type change if there is a biochemical mutation?

Answer 5

cell can go from a prototroph to an auxotroph or vice versa
prototroph: can make their own stuff
auxotroph: need to import it from the outside

Question 6

why can mutations happen?

Answer 6

• spontaneous, happen in DNA replication
• can be induced by mutagens
  physical (UV, X ray, gamma ray)
  chemical (5-bromouracil)

Question 7

what are the different types of point mutations?

Answer 7

only one base pair changes
- silent (does not change the codon)
- missense (changes the codon)
- nonsense (creates a stop codon)
- frameshift (deletions and insertions)

Question 8

what is a forward and reversion mutation?

Answer 8

forward mutation= starting from the reference wild type
reversion mutation= back to wild type phenotype, but that doesn’t necessarily mean that the sequence is the same
- original wild type sequence
- new codon but same AA (silent)
- new codon but similar AA (missense)

Question 9

where can a suppressor mutation be?

Answer 9

• intragenic: same gene but another location
• extragenic: different location

Question 10

what are some mistakes that can be made by DNA polymerase 3 and what does that lead to?

Answer 10

1. slippage in the new strand
   leads to an addition in the next round of replication
2. slippage in the parental strand
   leads to a deletion in the next round of replication

Question 11

how is the thymine dimer created and what is it?

Answer 11

through exposure to UV, a covalent bond between thymines is created, and that creates a shape distortion of the DNA

Question 12

what is 5-bromouracil and how can it be incorporated into the DNA?

Answer 12

exists in two forms: keto and enol
the two forms are present in a 50:50 ratio
the keto form can bind to adenine
the enol form can bind to guanine
this means that 5-bromouracil can be incorporated into the sequence by DNA pol III
-> you can induce mutations with this

Question 13

what are mechanisms of DNA repair and what do they deal with?

Answer 13

1. proofreading activity of DNA polymerases (3’ to 5’ exonuclease activity) (can still make mistakes)
2. mismatch repair
   not complementary base pairs
3. nucleotide excision repair
   distorted DNA (thymine dimer)
4. base excision repair (backbone intact but base is missing)
   apurinic and apyrimidinic sites
   damaged or unnatural bases (RNA in DNA)

Question 14

how does mismatch repair work?

Answer 14

1. the MuS protein finds a mismatch
   the MutS/MutL complex is bound to MutH, which is already bound to a hemimethylated sequence
2. MutH makes a cut in the nonmethylated strand
3. exonuclease begins at this cleavage site and digest the nonmethylated strand beyond the base mismatch
4. DNA polymerase fills in the vacant region
5. DNA ligase seals the end and the mismatch has been repaired

Question 15

how does nucleotide excision repair work?

Answer 15

1. the UvrAB complex tracks along the DNA in search of damaged DNA
2. after damage is detected, UvrAB is released and UvrC binds
3. UvrC makes cuts on both sides of the thymine dimer
4. UvrD is a helicase that remove the damaged region
5. UvrB and UvrC are also released
6. DNA polymerase fills the gap and DNA ligase seals the gap

Question 16

how does base excision repair work?

Answer 16

1. DNA glycosylase recognises an abnormal base and cleaves the bond between the base and the sugar
2. AP endonuclease recognises a missing base and cleaves the DNA backbone on the 5’ side of the missing base
3. DNA polymerase uses its 5’->3’ exonuclease activity to remove the damaged region and then fills the region with normal DNA
4. DNA ligase seals the gap