Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative. Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.

To protect germ cells from genomic instability, surveillance mechanisms ensure meiosis occurs properly. In mammals, spermatocytes that display recombination defects experience a so-called recombination-dependent arrest at the pachytene stage, which relies on the MRE11 complex—ATM—CHK2 pathway responding to unrepaired DNA double-strand breaks (DSBs). Here, we asked if p53 family members—targets of ATM and CHK2—participate in this arrest. We bred double-mutant mice combining a mutation of a member of the p53 family (p53, TAp63, or p73) with a Trip13 mutation. Trip13 deficiency triggers a recombination-dependent response that arrests spermatocytes in pachynema before they have incorporated the testis-specific histone variant H1t into their chromatin. We find that deficiency for either p53 or TAp63, but not p73, allowed spermatocytes to progress further into meiotic prophase despite the presence of numerous unrepaired DSBs. Even so, the double mutant spermatocytes apoptosed at late pachynema because of sex body deficiency; thus p53 and TAp63 are dispensable for arrest caused by sex body defects. These data affirm that recombination-dependent and sex body-deficient arrests occur via genetically separable mechanisms.

Precise execution of recombination during meiosis is essential for forming chromosomally-balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these remain poorly understood, particularly in mammals. Here we report that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated resected DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.

Recombination rates vary in intensity and location at the species, individual, sex and chromosome levels. Despite the fundamental biological importance of this process, the selective forces that operate to shape recombination rate and patterns are unclear. Domestication offers a unique opportunity to study the interplay between recombination and selection. In domesticates, intense selection for particular traits is imposed on small populations over many generations, resulting in organisms that differ, sometimes dramatically, in morphology and physiology from their wild ancestor. Although earlier studies suggested increased recombination rate in domesticates, a formal comparison of recombination rates between domestic mammals and their wild congeners was missing. In order to determine broad-scale recombination rate, we used immunolabeling detection of MLH1 foci as crossover markers in spermatocytes in three pairs of closely related wild and domestic species (dog and wolf, goat and ibex, and sheep and mouflon). In the three pairs, and contrary to previous suggestions, our data show that contemporary recombination rate is higher in the wild species. Subsequently, we inferred recombination breakpoints in sequence data for 16 genomic regions in dogs and wolves, each containing a locus associated with a dog phenotype potentially under selection during domestication. No difference in the number and distribution of recombination breakpoints was found between dogs and wolves. We conclude that our data indicate that strong directional selection did not result in changes in recombination in domestic mammals, and that both upper and lower bounds for crossover rates may be tightly regulated.

Whole-genome doubling (WGD) is common early in tumorigenesis. WGD doubles ploidy and centrosome number. In the ensuing mitoses, excess centrosomes form a multipolar spindle, resulting in a lethal multipolar cell division. To survive, cells must cluster centrosomes to allow bipolar cell division. Cancer cells are often more proficient at centrosome clustering than untransformed cells, but the mechanism behind increased clustering ability is not well understood. Heterozygous missense mutations in PPP2R1A, which encodes the alpha isoform of the ''scaffolding'' subunit of PP2A (PP2A-Aa), positively correlate with WGD. We introduced a heterozygous hotspot mutation, P179R, into PPP2R1A in human RPE-1 cells. PP2A-Aa P179R decreases PP2A assembly and intracellular targeting in mitosis. Strikingly, PP2A-Aa P179R enhances centrosome clustering when centrosome number is increased either by cytokinesis failure or centrosome amplification, likely through PP2A-Aa loss of function. Thus cancer-associated mutations in PP2A-Aa may increase cellular fitness after WGD by enhancing centrosome clustering.