Lecture 3

Question 1

Point mutations definition

Answer 1

Mutations at a single point (most often a single base pair)
in a genome - SNPs (single nucleotide polymorphisms)

Question 2

3 main categories of mutations

Answer 2

• point mutations
• substitution / indels (insertion or deletion of base-pairs)
• can also be deletion (loss) or large-scale rearrangements or insertion of mobile genetic elements

Question 3

Many point mutations may occur____

Answer 3

Spontaneously i.e with no evident external cause

Question 4

How do spontaneous mustions occur?

Answer 4

• require DNA synthesis
• can involve “mistakes” by DNA polymerase, the enzyme that synthesises DNA
• can arise through tautomerisation of bases - a base transiently flips into a different isomer that has different base-pairings properties (SNP result from Tautomerisation?)

Question 5

Tautomerisation of bases affects ____-_____

Answer 5

Base-pairing

Question 6

Example of normal base-pairs vs tautomeric base pairing

Answer 6

Base - changes structure - rearranges - giving cytosine a different form - imino form - this makes it much more chemically favourable to match to adenine

• same for thymine

Question 7

How does the bacteria cut down the rate of spontaneous mutation

Answer 7

• DNA polymose ‘ checks’ each new base-pair
• if it has made a mistake it backs up and removes the incorrect base (over 99% effective)
• DNA synthesis then resumes

Question 8

What serves as a prof reading function and how does it do this?

Answer 8

Both DNA pol 1 and DNA pol 111 serve a “proofreading” function by excising incorrectly inserted mismatched bases

• once the mismatched base is removed, the polymerase has another chance to add the correct complementary base

Question 9

What is the importance of proof reading?

Answer 9

DNA proof-reading reduces the frequency of spontaneous mutation in bacteria

• in mice, lack of DNA proofreading causes increased mutations in mitochondrial DNA and pre mature aging of mice

Question 10

Two ways of reducing spontaneous mutations

Answer 10

• proof reading by DNA pol 1 and 111
• the mismatch repair system

Question 11

Properties of the mismatch repair system

Answer 11

• can detect non-matched “pairs” of bases in DNA molecules
• can determining which of the bases is wring
• can remove the wrong base

Question 12

MMR (mismatch repair) process

Answer 12

• MutS protein detects mismatches of newly replicated DNA
• The binding of MutS to distortions in the DNA double helix recruits MutL and MuTH
• MutH cuts the newly synthesised strand containing the incorrect base

(- without the ability to discriminate between the parental and newly synthesised strands, the MMR system could not determine which base to excise - hoe does MutH know which is wrong? )

Question 13

How does MMR (MutH) know which strand is newly synthesised (to fix)?

Answer 13

• Strand recognition by MutH is directed by adenine methylation at GATC sequences
• becuase adenine methylation occurs after DNA synthesis, newly synthesised DNA is temporarily unmodified, an this temporary absence of methylation directs repair to the new strand

DNA inside E.coli is chemically modified by methylation of adenine bases in the DNA - newly synthesised strands will not have a mathylated base - thus MutH can recognise it as the one to be fixed

Does not affect the pairing of the bases with thymines

Question 14

What can the MMR system identify?

Answer 14

• system can identify mismatches AND
• distinguish the original (correct) and new (incorrect) strand of the DNA molecule

Question 15

How MutS, MutH and Mut L repair in MMR

Answer 15

• MutS protein detects mismatches of newly replicated DNA
• The binding of MutS to distortions in the DNA double helix recruits MutL and MuTH
• Strand recognition by MutH is directed by adenine methylation at GATC sequences
• becuase adenine methylation occurs after DNA synthesis, newly synthesised DNA is temporarily unmodified, an this temporary absence of methylation directs repair to the new strand
• the MutH endonuclease cuts the unmethylated strand
• MutS - MutL also activates excision, which involves the DNA helicase UvrD and exonucleases
• DNA polymerase 3 repairs the single stranded gap, and DNA ligaments generates a continuous covalent DNA back bone

Question 16

Two ways ? Mismatches can arise?

Answer 16

• mistakes in DNA replicaigton
• spontaneous chemical changes to bases

Question 17

How mismatches arise form spontaneous chemical changes to bases

Answer 17

• Deamination, the hydrolytic removal of an amine group
  E.g deamination converts cytosine to uracil
• Uracil base pairs with adenine in replication, converting a C-G base pair into a T-A base pair

Question 18

What are indel mutations

Answer 18

• insertiaons or deletions of one or more base pairs
• caused by DNA replication errors

Question 19

How do indel mutations arise?

Answer 19

Through an error during DNA replication
- DNA “slippage” results in loss or incorporation of a new base-pair into the DNA

Indels arise when loops in single stranded regions of DNA are stabilised by the “slipped mispairing” of repeated sequences in the course of DNA replication

Question 20

Indel mutation - insertion diagram vs deletion diagram

Answer 20

• deletion occurs in parental strand

Question 21

What are mutations?

Answer 21

Changes to the sequence of base-pairs in DNA molecules

Question 22

Two main ways that mustaions arise spontaneously

Answer 22

• tautomerisation
• indel mutation

Question 23

Two main mechanisms to reduce the frequency of mutaions

Answer 23

• DNA proof reading
• mismatch repair system MMR

Question 24

Rare base pairing - tautormisiration - why it martches better with the ‘wrong’ base

Answer 24

Hydrogen bonding?

Question 25

How base tautomers cause mutaion

Answer 25

? Don’t get this?

Question 26

Answer 26

Question 27

If u need a real world example - include TB and medication

Answer 27

- TB becomes resistance to medication cos it keeps mutating