Molecular pathways of meiotic prophase checkpoints in mice

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Halving of chromosome numbers in meiosis is the hallmark of the lifecycle of sexually-reproducing organisms. Meiotic segregation of chromosomes requires that homologous copies of each chromosome (homologs) form physical connections in meiotic prophase. In most taxa including mammals, these connections depend on chromosomal crossovers produced by recombination. Programmed formation of DNA double-strand breaks (DSBs) initiates meiotic recombination. Meiotic DSBs result in single stranded DNA (ssDNA) ends which invade homologs promoting chromosomal synapsis. In turn, synapsis promotes recombination-mediated DSB repair and generation of crossovers.To protect genome integrity in meiosis, quality control mechanisms/checkpoints ensure that all homolog pairs synapse, and that DSBs, which are potentially genotoxic, are repaired before meiocytes exit prophase. A central unresolved question is whether the DNA-damage checkpoint carries out the quality control for both DSB repair and synapsis or if distinct checkpoints monitor DSB repair and synapsis. This question is rooted in a debate on the role of HORMAD2, which we identified as a critical component in meiosis-specific quality control of recombination. HORMAD2 preferentially binds to chromosomal regions where synapsis has not formed yet. Unrepaired DSBs are also concentrated in these regions.Based on our data we hypothesize that HORMAD2 recruits ATR to unsynapsed chromosomal regions, and establishes a synapsis checkpoint that does not require DSBs to trigger elimination of synapsis-deficient meiocytes. In an alternative hypothesis, HORMAD2 blocks repair of DSBs to activate a DNA-damage checkpoint in unsynapsed regions instead of, or in addition to, a synapsis checkpoint. Verdict on these two models was hindered by three main factors. First, proper meiotic DSB repair and chromosomal synapsis are mutually dependent, making it difficult to separate checkpoint activation by defects in synapsis and/or DSB repair. Second, it is unclear if DSB-independent binding of HORMAD2-ATR complex to unsynapsed regions can establish a checkpoint. Third, standard methods, which rely on marker proteins to monitor DSB repair, failed to conclusively answer if HORMAD2 blocks DSB repair until proper synapsis is formed.We developed two new mouse models and a new method which overcome the main impediments in meiotic checkpoint analysis: (1) a mouse model that separates DSB repair and synapsis defects (factor 1), (2) a mouse model where HORMAD2 fails to bind chromosomes (factor 2), and (3) a method for the direct detection of meiotic ssDNA ends instead of protein markers of recombination (factor 3). Using these tools we will decisively test current models for meiosis quality control, revealing fundamental features of genetic inheritance with implication for human reproduction health.