Abstract
During gamete formation, crossover recombination must occur on replicated DNA to ensure proper chromosome segregation in the first meiotic division. We identified a Mec1/ATR- and Dbf4-dependent replication checkpoint in budding yeast that prevents the earliest stage of recombination, the programmed induction of DNA double-strand breaks (DSBs), when pre-meiotic DNA replication was delayed. The checkpoint acts through three complementary mechanisms: inhibition of Mer2 phosphorylation by Dbf4-dependent Cdc7 kinase, preclusion of chromosomal loading of Rec114 and Mre11, and lowered abundance of the Spo11 nuclease. Without this checkpoint, cells formed DSBs on partially replicated chromosomes. Importantly, such DSBs frequently failed to be repaired and impeded further DNA synthesis, leading to a rapid loss in cell viability. We conclude that a checkpoint-dependent constraint of DSB formation to duplicated DNA is critical not only for meiotic chromosome assortment, but also to protect genome integrity during gametogenesis. DOI:http://dx.doi.org/10.7554/eLife.00844.001.
Highlights
During meiosis, a single round of DNA replication is followed by two nuclear divisions to produce haploid gametes from diploid progenitor cells
To determine whether double-strand breaks (DSBs) formation is coordinated with bulk DNA replication during meiotic S phase (meiS) in budding yeast, we exposed cells to increasing doses of the replication inhibitor hydroxyurea (HU) and measured the kinetics of DNA replication and DSB formation
Quantification of FACS profiles and Southern analysis revealed that DSBs appeared just after bulk DNA replication was completed (4C DNA content appeared) in 0 or 5 mM HU samples, and were fully suppressed when replication was blocked by 20 mM HU (Figure 1C)
Summary
A single round of DNA replication is followed by two nuclear divisions to produce haploid gametes from diploid progenitor cells. DNA exchanges between homologs are the result of the repair of ∼160 DNA double-strand breaks (DSBs), which occur immediately following pre-meiotic S phase (meiS) and are distributed across all 16 chromosome pairs. DSB formation requires the phosphorylation of Mer by two cell cycle kinases, cyclin dependent-kinase (CDK) and the Dbf4-dependent Cdc kinase (DDK) (Henderson et al, 2006; Sasanuma et al, 2008; Wan et al, 2008). The complexity of this process reflects the fact that meiotic genome fragmentation needs to be carefully controlled to limit genome instability
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