Mismatch repair Flashcards
(35 cards)
1
Q
What does mismatch repair recognise and repair? (2)
A
- Single non Watson-Crick base pairs
- Small insertions/deletion loops (IDLs) of typically 1-3nt
2
Q
What processes are monitored by the mismatch repair pathway? (2)
A
- DNA replication
- Recombination between homeologous sequences
3
Q
What is homeologous recombination?
A
Recombination between similar but non-identical sequences
4
Q
What is the importance of mismatch repair? (2)
A
- Important for genome integrity
- Mutations cause HNPCC and also found in other sporadic cancers
5
Q
What is HNPCC?
A
Hereditary nonpolyposis colorectal cancer
6
Q
Which pathway deals with large DNA adducts?
A
Nucleotide excision repair
7
Q
How are IDLs formed? (4)
A
- Polymerase falls off and doesn’t reattach in the right place
- Often happens in microsatellite sequences as the polymerase doesn’t know which repeat to reattach to
- Either copies too many repeats or too few repeats
- Results in a loop structure in the daughter strand (insertion) or in the template strand (deletion)
8
Q
How does proofreading occur during DNA replication? (5)
A
- MMR is not proofreading as it only acts on mismatches that have escaped the proofreading process (happens afterwards)
- Polymerase stalls when it makes a mistake and dissociates
- Exonuclease removes the mismatched strand
- Most polymerases have an intrinsic proofreading activity in the form of exonuclease activity to ‘rewind’ and resynthesise the region correctly
- MMR works with the proofreading pathway very effectively (~1 error every 250 generations in S. cerevisiae)
9
Q
How does MMR occur in E. coli? (6)
A
- Replication error causes a mismatch
- MutS recognises the mismatch
- MutS attracts MutL and MutH
- MutH nicks the newly synthesised strand
- Exonucleolytic degradation past the mismatch
- Resynthesis
10
Q
How does a footprint analysis work? (5)
A
- Incubate DNA substrate with a purified protein (e.g. MutS) to allow binding
- Treat with low concentration of DNase endonuclease which should make 1 cut in each DNA molecule
- If the protein is bound to the DNA, the DNase won’t be able to cut it so there will be a gap in the banding pattern
- Look at the differences in activity between perfectly matched duplexes (homoduplex) and mismatched duplexes (heteroduplex)
- Mismatch = MutS binds = digestion by DNase blocked = gap in the ladder
11
Q
What is the structure of MutS bound to a mismatch in E.coli? (3)
A
- MutS homodimer wraps around the DNA mismatch
- Mismatch recognition domain creates a kink at the mismatched site (only 1 subunit binds the mismatch, asymmetry)
- ATPase domain binds ATP once a mismatch has been found
12
Q
How does MutS recognise heteroduplex DNA? (4)
A
- Mismatches are sensed by changes in thermal stability as interactions between mismatched base pairs are weaker
- MutS binds non-specifically to DNA
- Conformational changes create a kink in the duplex to ‘test’ the thermal stability: mismatched pairs are easier to break
- Specific interactions with the mismatched site lock MutS in place
13
Q
How does the MMR machinery know which strand to repair in E.coli? (4)
A
- DNA of some bacteria is methylated at GATC sites (adenine) by Dam methylase
- Dam methylase is ~2 minutes behind the replication fork so for a while DNA is hemimethylated (only 1 strand while the new one is being made)
- Therefore in mismatched DNA it is the new unmethylated strand that needs to be nicked and repaired
- The MMR machinery needs to diffuse away from the mismatch to find a GATC site and work out which strand to repair
14
Q
How does the structure of MutS change after mismatch recognition? (4)
A
- Following mismatch recognition, MutS conformation changes to a sliding clamp that can move along the DNA
- Might travel a few hundred bp to find a GATC
- MutS dimer is 600x more stable on the DNA in the sliding clamp conformation than when MutS is searching for mismatches
- ATP hydrolysis is needed for testing mismatches but is suppressed after recognition so ATP is bound stably to the ATPase domain in sliding clamp mode
15
Q
What is the role of MutL? (4)
A
- Also forms a homodimer ring around the DNA
- Contacts with mismatch-bound MutS
- Interacts with MutH, UvrD and exonucleases
- Additional catalytic roles in eukaryotic homologues (endonuclease activity to perform the role of MutH)
16
Q
What is the role of MutH in MMR? (3)
A
- Endonuclease
- Nicks the UNmethylated DNA strand at a hemimethylated GATC site
- Requires Mg2+ binding, hemimethylated GTAC and MutL interaction
17
Q
What is the role of UvrD in MMR? (2)
A
- Helicase
- Unwinds the DNA from the nick back towards the mismatch
18
Q
What is the role of exonucleases in MMR? (2)
A
- At least 4 different ones degrade the newly synthesised strand
- Both 5’-3’ and 3’-5’ so it doesn’t matter which side of the mismatch the nick is on
19
Q
What are the final steps of MMR? (2)
A
- DNA polymerase resynthesises the region
- Nick is religated
20
Q
How do the E.coli MMR proteins interact? (3)
A
- MutS sliding clamp recruits MutL clamp which works in a similar way
- MutL moves away and associates with MutH and UvrD
- Several MutS clamps can be recruited to the mismatch during this so can be several sets of dimers present on the DNA
21
Q
Which polymerases are used for leading and lagging strand replication in eukaryotes? (2)
A
- Polε leading strand replication
- Polα and Polδ lagging strand replication
22
Q
How does eukaryotic MMR differ from E.coli? (2)
A
- More complex with more proteins
- No eukaryotic MutH homologue
23
Q
What are the MutS eukaryotic homologues? (4)
A
- MSH proteins (Mut S Homologues)
- MSH2
- MSH3
- MSH6
24
Q
What does a gelshift assay show? (3)
A
- Incubate DNA substrate with protein of interest
- If protein binds the MW of the DNA+protein increases and runs slower so you see a single band higher up in the gel
- Prominent band at the bottom represents unbound DNA
25
What MSH complexes form at mismatch sites and how does this compare to E.coli? (4)
- None of the MSH proteins bind DNA on their own, Msh2 binds ATP and pairs with Msh3 or Msh6 depending on the type of mismatch
- MSH2+MSH3 dimer binds larger IDLs and is equivalent to MutSβ
- MSH2+MSH6 dimer binds single bp mismatch or 1bp IDL and is equivalent to MutSα
- Heterodimers reflect the asymmetry of the bacterial homodimer
26
What are the MutL eukaryotic homologues? (3)
- MLH proteins (Mut L Homologues) and PMS proteins (Post Meiotic Segregation)
- MLH1+PMS2 (human, PMS1 in yeast) = MutLα, mostly for post-replicative MMR
- MLH1+MLH3 mostly for regulation of meiotic recombination
27
How does the activity of MutL eukaryotic homologues differ from in E.coli? (4)
- Many eukaryotic MutL homologues are not just matchmakers, also endonuclease activity
- E.g. MutLα is an endonuclease
- Therefore MutL homologues take the role of E.coli MutH: endonucleolytic cleavage of one strand in the heteroduplex DNA
- No sequence specificity for this activity unlike MutH
28
How is strand discrimination done in eukaryotic MMR? (2)
- yCdc9 (human DNA ligase 1) closes the nicks in newly synthesised DNA
- Temporal control during S phase so for a window of time there are lots of nicks in the new strand which is a way to tell which is the template strand and which is newly made
29
What are the 3 redundant pathways in eukaryotic MMR downstream from mismatch identification?
- Exo1 5'-3' exonuclease degrades the mistake strand which is followed by resynthesis, however still get good MMR in vivo KO of Exo1 so must be other pathways
- Rad27/FEN1 flap endonuclease coordinates resynthesis with Polδ then cleaves off the mismatched flap
- Another not well understood pathway which involved additional cleavage activity by MLH1
30
How is MMR involved in cancer? (5)
- 30% of colorectal cancer cases are due to an inherited mutation
- Mutations in MMR genes cause HNPCC (Lynch syndrome) which make up 2-4% of all colorectal cancer cases, individuals also susceptible to endometrial and ovarian cancer
- MMR-deficient cells are prone to microsatellite instability which is a hallmark of HNPCC
- This causes increased risk of acquiring tumorigenic mutations
- MMR is lost in 10-40% of sporadic cancers
31
How is MMR linked to recombination? (4)
- S. typhinurium and E. coli genomes are 3% diverged
- Measured conjugation (i.e. integration by recombination)
- Observed 735-fold more recombination in the absence of MutS meaning that MMR blocks recombination between these divergent sequences (homeologous recombination)
- MMR machinery can recognise the formation of a heteroduplex upon strand invasion and process it
32
What is the effect of MMR on recombination with non-homologous templates? (4)
- MMR limits recombination between homeologous DNA molecules
- A single mismatch can be enough to reduce recombination efficiency in MMR+ cells
- Reduction in recombination efficiency as sequence divergence increases (log-linear relationship)
- Same general pattern in MMR- cells although the overall recombination rate is higher
33
How does the MMR machinery limit recombination between homeologous templates?
- Recognition of mismatches
- Heteroduplex rejection
34
How does MMR recognition of mismatches limit recombination? (2)
- MMR machinery recognises mismatches in recombination intermediates e.g. D loop
- 'Traps' intermediates to stop them being resolved
35
What is heteroduplex rejection? (4)
- Unwinding of the mismatched DNA by helicases which aren't necessarily part of the post-replicative MMR machinery (antirecombination)
- MutS homologues attract 5'-3' helicases
- E. coli: UvrD
- S. cerevisiae: including Sgs1 and Srs2 (not involved in post-replicative MMR but important in other repair pathways)
