High Throughput Sequencing Reveals Alterations in the Recombination Signatures with Diminishing Spo11 Activity

摘要

Spo11 is the topoisomerase-like enzyme responsible for the induction of the meiosis-specific double strand breaks (DSBs), which initiates the recombination events responsible for proper chromosome segregation. Nineteen PCR-induced alleles of SPO11 were identified and characterized genetically and cytologically. Recombination, spore viability and synaptonemal complex (SC) formation were decreased to varying extents in these mutants. Arrest by ndt80 restored these events in two severe hypomorphic mutants, suggesting that ndt80 -arrested nuclei are capable of extended DSB activity. While crossing-over, spore viability and synaptonemal complex (SC) formation defects correlated, the extent of such defects was not predictive of the level of heteroallelic gene conversions (prototrophs) exhibited by each mutant. High throughput sequencing of tetrads from spo11 hypomorphs revealed that gene conversion tracts associated with COs are significantly longer and gene conversion tracts unassociated with COs are significantly shorter than in wild type. By modeling the extent of these tract changes, we could account for the discrepancy in genetic measurements of prototrophy and crossover association. These findings provide an explanation for the unexpectedly low prototroph levels exhibited by spo11 hypomorphs and have important implications for genetic studies that assume an unbiased recovery of prototrophs, such as measurements of CO homeostasis. Our genetic and physical data support previous observations of DSB-limited meioses, in which COs are disproportionally maintained over NCOs (CO homeostasis). Author Summary Most eukaryotes depend on the meiotic division to segregate each pair of chromosomes properly into their gametes. Chromosome segregation mistakes happening during meiosis are responsible for most miscarriages as well as many diseases such as Down's and Kleinfelter's syndromes in humans. Proper chromosome segregation during meiosis depends on efficient and regulated recombination events that link homologous chromosomes prior to the first meiotic division. These linkages are initiated at double-stranded breaks (DSBs) in chromosomal DNA by Spo11 and associated proteins. We isolated a valuable new set of SPO11 alleles in yeast with a wide range of Spo11 activity. Genetic analysis and high throughput sequencing of tetrads from these mutants has revealed unexpected features of meiotic recombination. First, Spo11 DSBs likely continue to form throughout a pachytene arrest in cells compromised for Spo11 activity. Second, the number of recombination initiation events in a given meiosis influences the repair outcome of those events. In addition, our results provide support for crossover homeostasis – a phenomenon in which crossovers are disproportionately maintained over other types of repair in the face of a decrease in DSBs.