Homologous recombination: resolution Flashcards
(21 cards)
1
Q
How do the products of HR differ in mitosis and meiosis? (3)
A
- Double Holliday junctions need to be resolved to form crossovers during meiosis to generate genetic diversity and ensure accurate homolog segregation
- Non-crossover formation is favoured in mitosis to avoid potential LOH and high levels of sister chromatid exchanges
- Crossover is avoided in mitosis either by use of anti-recombinogenic pathways (SDSA) or by actions of enzymes that promote dissolution (BLM helicase) rather than resolution
2
Q
Which HR pathways avoid crossover? (3)
A
- Synthesis-dependent strand annealing (avoids double Holliday junctions)
- Dissolution
- One pathway of resolution
3
Q
What is required for synthesis-dependent strand annealing? (3)
A
- Needs yeast Srs2 function (DNA helicase and DNA-dependent ATPase) which pushes the D loop across the DNA until it gets dissolved
- Disassembles Rap51p nucleoprotein filaments during recombination
- Functional homolog of human RTEL1
4
Q
What is synthesis-dependent strand annealing? (4)
A
- Strand invasion and DNA synthesis occurs using the homologous template sequence
- Srs2 unwinds the invading strand and disassembles the D loop, allowing it to reanneal to its original strand
- DNA synthesis occurs in the other strand using the newly made section of the complimentary strand as the template and is also reannealed
- Results in no crossover
5
Q
What are the 3 mechanisms for processing recombination intermediates (Holliday junctions)?
A
- Dissolution which is non-crossover
- 2 pathways for resolution which can result in crossover or non-crossover
6
Q
What are the 2 pathways for resolution?
A
- SLX-MUS
- GEN1
7
Q
What is dissolution? (5)
A
- Branch in the Holliday junction can migrate in either direction by breaking old base pairs and forming new ones via branch migration
- In a double Holliday junction they will migrate towards each other
- Sgs1/hBLM helicase pushes the Holliday junctions together (requires energy from ATP hydrolysis), creating tension in the duplexes (hemi-catenation)
- TopIII creates nicks in the DNA strands which reanneal to their original strands
- Results in no crossover
8
Q
What is Top3p? (2)
A
- DNA topoisomerase III
- Resolves DNA hemi-catenation where one single strand from one dsDNA duplex is interlaced with another single strand from another ds duplex, resulting in 2 separate duplexes
9
Q
What are Bloom and Werner syndromes? (2)
A
- Inherited syndromes that cause genomic instability and cancer predisposition
- BLM and WRN are part of the RecQ family of DNA helicases and human homologs of Sgs1
10
Q
What are the 2 outcomes for Holliday junction resolution by HJ resolvases?
A
- Cutting horizontally (the tangled strands) results in non-crossover
- Cutting vertically (the untangled strands) results in crossover
11
Q
What are Holliday junction resolvases? (3)
A
- Group of structure-selective endonucleases which exhibit a preference for binding and cleaving four-way DNA junctions
- Cleave HJ to generate 2 unconnected DNA duplexes
- Dimeric enzymes with twin active sites to catalyse 2 coordinated incisions within a single protein-HJ complex
12
Q
How do prokaryotic HJ resolvases work? (6)
A
- Prokaryotic HJ resolvase is a 3 subunit complex e.g. RuvABC
- RuvA specifically binds four-way DNA junctions and recruits RuvC to Holliday junction
- RuvB DNA helicase interacts with RuvA
- RuvA and RuvB promote branch migration
- RuvA and RuvB recruit RuvC to HJ and RuvC may displace RuvA to gain access
- RuvC forms a dimer and cuts the junction to form 2 duplex products
13
Q
What are the 2 pathways for HJ resolvases in eukaryotes?
A
- SLX1-MUS complex
- Yen1 (yeast ortholog of human GEN1)
14
Q
How was hGEN1/yYEN1 found in eukaryotes? (3)
A
- Used a HeLa nuclear extract, pulled out proteins bound to ssDNA and incubated them with cruciform DNA substrate
- Smaller product shows the structure has been resolved
- Identify protein responsible (GEN1) by mass spectrometry
15
Q
How does GEN1 work? (2)
A
- Performs 2 symmetrical cuts in the DNA junction
- 50% chance of crossover or non-crossover
16
Q
How was the SLX-MUS complex discovered? (5)
A
- Supercoiled plasmid containing a Holliday junction-like cruciform structure and incubated with potential resolvases
- Location of cut determines the product formed
- Symmetrical cuts (like GEN1) results in linear product
- Incubating with SLX1-4 proteins results in a nicked circular product, suggesting that SLX performs the initial cut (not efficient on its own)
- Adding MUS to SLX results in linear product suggesting both are required in this complex for HJ resolution
17
Q
How does the SLX-MUS complex work? (4)
A
- SLX-MUS is a HJ resolvase that coordinates the active sites of 2 distinct endonucleases during HJ resolution
- In response to CDK-mediated phosphorylation (dependent), SLX1-SLX4 - MUS81-EME1 associate at the G2/M transition (specific) to form a stable SLX-MUS holoenzyme
- EME1 is phosphorylated
- SLX makes the first nick and the complex of SLX-MUS makes the second nick
18
Q
How do phosphorylated proteins run compared to non-phosphorylated?
A
Appear larger MW because they run slower (higher up)
19
Q
When in the cell cycle are the SLX-MUS and GEN1 pathways active? (3)
A
- Mus81-Mms4 is inactive in S phase but then phosphorylation of Mms4 by CDK/Cdc5 causes activation in G2/M phase
- Yen1 is phosphorylated by CDKs and inactive during S phase but then dephosphorylation by Cdc14 causes nuclear localisation and activation during anaphase
- Distinct timing of activation
20
Q
Why does yYEN1 require phosphorylation for nuclear localisation but hGEN1 doesn’t? (3)
A
- Gen1 is usually excluded from the nucleus to prevent premature HJ resolution and competition with break induced repair (causes non-crossover) which would cause more crossover events
- Gen1 can access the DNA in mitosis when the nuclear envelope is dissolved during prophase which ensures any leftover chromosome bridges are cleaved before segregation
- In yeast the nucleus remains intact for mitosis so Yen1 requires phosphorylation to enter
21
Q
How are HJ resolution pathways controlled by the cell cycle? (4)
A
- S phase: Top3 and BLM are active to promote dissolution whereas resolution pathways cleave persistent double/single HJs and other secondary structures
- G2/M: resolvases are controlled by CDK1 phosphorylation (SLX-MUS)
- Any remaining HJ constructs are cleaved by GEN1 in anaphase
- All 3 pathways are required for genome maintenance and to ensure all HJs are removed prior to chromosome segregation in mitosis
