Abstract
The Dbf4-dependent Cdc7 kinase (DDK) is essential for chromosome duplication in all eukaryotes, but was proposed to be dispensable for yeast pre-meiotic DNA replication. This discrepancy led us to investigate the role of the unstable Cdc7-regulatory protein Dbf4 in meiosis. We show that, when Dbf4 is depleted at the time of meiotic induction, cells enter the meiotic program but do not replicate their chromosomes. Surprisingly when Dbf4 is depleted after the initiation of DNA synthesis, S phase goes to completion, but most cells arrest before anaphase I. Deletion of the cohesin Rec8 suppresses this phenotype, suggesting a distinct role of DDK for meiotic chromosome segregation. As after Cdc5 depletion, a fraction of cells undergo a single equational division suggesting a failure to mono-orient sister kinetochores. Our results demonstrate that Dbf4 is essential for DNA replication during meiosis like in vegetative cells and provide evidence for an additional role in setting up the reductional division of meiosis I.
Highlights
Despite its well characterized function for DNA replication in the mitotic cell cycle, little is known on the role of dependent Cdc7 kinase (DDK) during meiosis
The present study demonstrates that Cdc7-Dbf4 is required during the early stages of meiosis to initiate DNA replication
These data contrast with earlier findings that suggested that CDC7 was not required for pre-meiotic DNA replication
Summary
Cells were grown in YEP medium (1% yeast extract, 2% bacto-peptone) supplemented with 50 mg of adenine per liter and 2% dextrose (YEPD). Cells were washed twice in water and incubated in SPM with vigorous shaking at 30 °C. Centrifugal Elutriation—Cells were grown as described above into one liter of YPA to a concentration of ϳ2 ϫ 107 cells/ml. Elutriated G1 cells were collected, washed twice in water, and dissolved in SPM to a concentration of ϳ3 ϫ 107 cells/ml and incubated at 30 °C with vigorous shaking. Protein extracts were prepared from trichloroacetic acid-fixed cells, and analyzed by Western blot as described previously [10]. Cells were washed twice with potassium phosphate buffer (KPO4) and resuspended in 100 l of phosphate-buffered saline, pH 7.5, containing 4Ј,6-diamidino-2-phenylindol (DAPI) and incubated for at least 30 min. The amount of replicated DNA was estimated measuring the re-emergence from the gel wells of fully replicated chromosomes from nine representative chromosomal bands, and the average value was plotted on a graph considering the diploid DNA content as 100%
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