Chemical Inactivation of Cdc7 Kinase in Budding Yeast Results in a Reversible Arrest That Allows Efficient Cell Synchronization Prior to Meiotic Recombination

Abstract

Genetic studies in budding yeast have provided many fundamental insights into the specialized cell division of meiosis, including the identification of evolutionarily conserved meiosis-specific genes and an understanding of the molecular basis for recombination. Biochemical studies have lagged behind, however, due to the difficulty in obtaining highly synchronized populations of yeast cells. A chemical genetic approach was used to create a novel conditional allele of the highly conserved protein kinase Cdc7 (cdc7-as3) that enables cells to be synchronized immediately prior to recombination. When Cdc7-as3 is inactivated by addition of inhibitor to sporulation medium, cells undergo a delayed premeiotic S phase, then arrest in prophase before double-strand break (DSB) formation. The arrest is easily reversed by removal of the inhibitor, after which cells rapidly and synchronously proceed through recombination and meiosis I. Using the synchrony resulting from the cdc7-as3 system, DSB-dependent phosphorylation of the meiosis-specific chromosomal core protein, Hop1, was shown to occur after DSBs. The cdc7-as3 mutant therefore provides a valuable tool not only for understanding the role of Cdc7 in meiosis, but also for facilitating biochemical and cytological studies of recombination.