Homologous recombination: meiotic recombination Flashcards
(35 cards)
What are the models for DSB repair following resection and strand invasion/extension? (3)
- Break induced replication (BIR) (non-crossover)
- Normal DSB repair creating a dHJ, either dissolution (non-crossover) or resolution (non-crossover OR crossover)
- Synthesis-dependent strand annealing (SDSA) (non-crossover)
What is the purpose of HR in mitotic cells?
Repair spontaneous DSBs and maintain genomic stability
What is the source of DSBs in mitotic cells?
Spontaneous or from an exogenous agent
What are the consequences of HR in mitotic cells? (4)
- Usually non-crossover
- Usually uses sister chromatid (identical) as the template
- Uses Rad51 recombinase
- Usually uses SDSA or HJ dissolution pathways to prevent crossover events
What is meiosis?
Cell division process which results in 4 daughter cells, each with half the number of chromosomes of the parent cell
When during meiosis does crossing over occur?
Prophase I
How does HR happen during meiosis? (3)
- Prophase I: chromosomes condense and homologous chromosomes pair up to form bivalent chromosomes (synapsis)
- Programmed DSBs occur due to expression of a specific nuclease (Spo11) to initiate HR using the homologous chromosome as the template, resulting in chiasmata and crossover events
- 2 rounds of segregation result in 4 genetically unique haploid daughter cells
What is synapsis? (2)
- Pairing up of homologous chromosomes during meiosis
- Align on top of each other to form the synaptonemal complex which allows HR to use homologous chromosomes as the template DNA
What is the importance of crossing over in meiosis? (2)
- Generates genetic diversity and drives evolution
- Ensures accurate chromosome segregation at the first meiotic division because the crossover is a tether between homologs which ensures proper alignment at the metaphase plate and correct spindle attachment
What are the characteristics of the breaks formed in meiosis crossover? (6)
- Distributed non-randomly throughout the genome and concentrated within distinct hotspot regions of chromosomes, typically ~50-300bp
- Chromatin accessibility determines hotspot locations e.g. nucleosome-depleted regions at promoters
- Chromosome size is a factor as smaller chromosomes have higher DSB densities
- DSBs are suppressed near telomeres and centromeres (important regions)
- Chromosomal domains with higher/lower DSB frequency alternate which positively correlates with GC content
- Hotspots tend to be AT-rich and flanked by sequences enriched for H3K4me3 marker (transcriptionally active)
Why is the mobility of meiotic DSB DNA faster on an agarose gel when pronase is added?
Meiotic DSB DNA runs faster with pronase which suggests that meiotic DNA DSB ends are covalently bound to protein i.e. topoisomerases (Spo11 which is a topoisomerase-like protein)
What was the evidence for Spo11 covalently binding to meiotic DSBs? (2)
- Immunoprecipitation of HA-tagged Spo11 which co-precipitates DSB DNA
- Yeast experiment but Spo11 is conserved in humans
How does Spo11 work? (4)
- Spo11 binds and cleaves dsDNA to create DSB
- After the cleavage a tyrosine in Spo11 forms a covalent bond with the 5’ end phosphate group at both ends of the DNA break
- Spo11 released from the chromatin by an endonucleolytic cleavage reaction mediated by MRX and Sae2 which liberates Spo11 attached to a short oligonucleotide
- 5’ strands are further resected by 5’-3’ exonucleases (Exo1 in yeast) to produce long single stranded tails which are coated with RPA
Which complexes are involved in Spo11 cleavage and repair of the DSB? (3)
- Core complex
- MRX
- RMM
What does the core complex do? (2)
- Contains Spo11
- Performs the cut to create the DSB
What does the MRX (hMRN) complex do? (3)
- Contains Mre11, Xrs2 and Rad50
- Resects the break site and causes release of Spo11(attached to a short oligonucleotide) via an endonucleolytic cleavage reaction mediated by MRX and Sae2
- Only been reported in budding yeast and C.elegans, unkown if required in mice
What does the RMM complex do? (2)
- Contains Mei4
- May be involved in promoting the process but not understood how
What is Mei4? (2)
- Rec114, Mei4 and Mer2 (RMM) are a subgroup of functionally conserved DSB proteins
- Important for meiotic DSB formation but function not completely understood
How is meiotic DSB formation restricted to prophase I? (9)
- Tied to cell cycle control
- Replication positively impacts DSB formation by promoting Mer2 phosphorylation
- Replication stress inhibits DSB formation through Mec1 (checkpoint kinase) activation (balance)
- Recombination defects activate Mec1 which extends prophase by preventing Ndt80 activation (positive feedback loop)
- Ndt80 inhibits formation of DSBs
- Activation of DDR kinase Tel1 inhibits further DSB formation (negative feedback loop)
- Hotspot competition (Tel1-independent) and DSB interference (Tel1-dependent) impact spatial distribution of DSB formation which limits coincident formation of 2 DSBs
- Homolog engagement shuts down DSB formation through SC-dependent removal of DSB proteins
- Exit of pachytene following Ndt80 activation
How does strand invasion occur in meiosis DSB repair?
RPA is replaced by recombinases Rad51 and Dmc1 that form a nucleoprotein filament and search for sequence similarity preferentially located on the homologous chromosome, producing D loop structures
How does strand invasion differ between mitosis and meiosis?
Rad51 is facilitated by Rad52 and BRCA2 in mitosis but in meiosis it uses Dmc1
What is Dmc1? (3)
- Meiosis-specific recombinase required for strand invasion during meiosis
- Has similar biochemical properties to Rad51 (binds ssDNA and dsDNA, forms nucleoprotein filaments)
- Promotes inter-homolog repair (rather than sister chromatid template as in mitosis) but the mechanism is unclear
How does resolution of HJ’s differ between mitosis and meiosis? (3)
- Crossover is avoided during mitosis by using anti-recombinogenic pathways that disengage joint molecules at an early stage (SDSA) or by using enzymes that promote dHJ dissolution (BLM/Topo3alpha)
- HJ resolvases (SLX-MUS/GEN1) provide a safeguard mechanism to ensure that all HJs are removed prior to chromosome segregation
- Meiosis aims to introduce genetic diversity via crossover events
What is SDSA?
Synthesis-dependent strand annealing
