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要旨:
Meiotic gene conversion has a major impact on genome diversity. Both crossovers and non-exchange conversions cluster into distinct recombination-active regions that we call hotspots. Hotspot analysis in humans has focused on the description of crossover profiles and only few hotspots had been tested for crossover and non-exchange gene conversion. Therefore, very little was known about the frequency and distribution of non-exchange conversions and how well they correlate with crossing over. Six extremely active recombination hotspots were analysed by using small pool PCR approaches on sperm DNA to detect both types of recombinant molecules. Non-exchange conversions were detectable and arose at high frequencies (0.01-0.47%) per sperm, in addition to crossovers (0.2-0.70%). Conversion tracts were short and their distribution defined a steep conversion gradient, centred at the peak of crossover activity and probably marking the zone of recombination initiation. It was also observed that non-exchange gene conversion and crossover frequencies were positively correlated, not just between men at the same hotspot but equally when compared across several hotspots. On average, non-exchange gene conversions spanning a marker close to the centre of the hotspot occurred at 50% of the crossover frequency. Hotspot regulation in cis had been described previously to results in different initiation efficiencies between interacting haplotypes. Preferential initiation on a more active haplotype, in turn leads to the overtransmission of alleles from the less active haplotype. Additional hotspots that showed a phenomenon of biased gene conversion were described in this study, with crossovers and non-exchange gene conversions influenced to the same degree. More unusually, biased gene conversion specifically affecting non-exchange events was also observed at two hotspots. Here single SNP heterozygosities appear responsible for triggering the bias in cis. Crossovers were not affected, which may provide evidence for distinct crossover and non-crossover pathways operating at human hotspots. Inter-individual differences in recombination frequencies between men at a given hotspot established PRDM9 as major trans-regulator of hotspot activity. PRDM9 regulation was characterised at two hotspots activated by specific sets of PRDM9 variants. At both hotspots a sequence motif, proposed to be the PRDM9 binding site in vitro, was not found to be responsible for hotspot activation as had been predicted previously. Curiously, better motif matches were not correlated with higher crossover frequencies, and PRDM9 can in fact activate hotspot without the proposed binding motif.
