An essential feature of meiosis is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks (DSBs) is repaired using an intact homologous non-sister chromatid rather than a sister. Involvement of Mec1 and Tel1, the budding yeast homologs of the mammalian ATR and ATM kinases, in meiotic interhomlog bias has been implicated, but the mechanism remains elusive. Here, we demonstrate that Mec1 and Tel1 promote meiotic interhomolog recombination by targeting the axial element protein Hop1. Without Mec1/Tel1 phosphorylation of Hop1, meiotic DSBs are rapidly repaired via a Dmc1-independent intersister repair pathway, resulting in diminished interhomolog crossing-over leading to spore lethality. We find that Mec1/Tel1-mediated phosphorylation of Hop1 is required for activation of Mek1, a meiotic paralogue of the DNA-damage effector kinase, Rad53p/CHK2. Thus, Hop1 is a meiosis-specific adaptor protein of the Mec1/Tel1 signaling pathway that ensures interhomolog recombination by preventing Dmc1-independent repair of meiotic DSBs.

Budding yeast Mec1, homolog of mammalian ATR, is an essential protein that mediates S-phase checkpoint responses and meiotic recombination. Elimination of Mec1 function leads to genomewide fork stalling followed by chromosome breakage. Breaks do not result from stochastic collapse of stalled forks or other incidental lesions; instead, they occur in specific regions of the genome during a G2 chromosomal transition. Break regions are found to be genetically encoded replication slow zones (RSZs), a newly discovered yeast chromosomal determinant. Thus, Mec1 has important functions in normal S phase and the genome instability of mec1 (and, analogously, ATR-/-) mutants stems from defects in these basic roles.

We have found that under appropriate conditions, an allele-specific polymerase chain reaction (PCR) can achieve a sensitivity suitable for measuring specific, infrequent mutations in single cell systems or in animal tissues. Using the 12th codon GC-to-AT mutation in the rat c-Ha-ras gene as a model system, we have defined conditions that allow for measurement of mutations present at frequencies as low as one in 10(5) gene copies. Our approach involved the use of PCR primers that created a single mismatch with the mutated allele (GAA) but created a double mismatch with the wild-type allele (GGA). Five out of the six such double-mismatch primers we tested permitted amplification of the mutant allele (GAA) with a high degree of specificity. The specificity of the assay was further enhanced by using a two-step PCR cycle consisting of a denaturation step (1 min incubation at 94 degrees C) and an annealing/extension step (1 min incubation at 50 degrees C) in the presence of 10% (vol/vol) glycerol. Reconstruction experiments using genomic DNA demonstrate that this procedure cna measure the presence of 30 copies of the transforming ras allele present amongst 3 x 10(6) copies of the wild-type allele.

An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or “DSB homeostasis”, might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.