Sic1-induced DNA rereplication during meiosis

Abstract

Orderly progression through meiosis requires strict regulation of DNA metabolic events, so that a single round of DNA replication is systematically followed by a recombination phase and 2 rounds of chromosome segregation. We report here the disruption of this sequence of events in Saccharomyces cerevisiae through meiosisspecific induction of the cyclin-dependent kinase (CDK) inhibitor Sic1 mutated at multiple phosphorylation sites. Accumulation of this stabilized version of Sic1 led to significant DNA rereplication in the absence of normal chromosome segregation. Deletion of DMC1 abolished DNA rereplication, but additional deletion of RAD17 restored the original phenotype. Therefore, activation of the meiotic recombination checkpoint, which arrests meiotic progression at pachytene, suppressed DNA rereplication resulting from Sic1 stabilization. In contrast to deletion of DMC1, deletion of NDT80, which encodes a transcription factor required for pachytene exit, did not inhibit DNA rereplication. Our results provide strong evidence that CDK activity is required to prevent inappropriate initiation of DNA synthesis before the meiotic divisions.checkpoint ͉ cyclin-dependent kinase ͉ premeiotic DNA replication ͉ recombination

Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

“…We have developed a system in which cells undergoing meiosis experience at least one extra round of DNA replication (Sawarynski et al 2009). This phenotype occurs upon deregulation of CDK activity through early meiosis-specific expression (via the HOP1 promoter) of SIC1 ∆ P HA , encoding a Sic1 HA variant lacking critical CDK phosphorylation sites that are necessary for its destruction (Verma et al 1997).…”

“…Construction of the HOP1pr-SIC1 ∆ P HA module, and its integration into the genome, were described previously (Sawarynski et al 2009). In most cases, deletion mutations were generated in haploids by homology-directed site-specific replacement with selectable markers (Baudin et al 1993).…”

“…All strains listed were constructed in the W303 background (Thomas and Rothstein 1989): diploid wild type = MAT a / α ade2-1/” ura3-1/” leu2-3,112/” his3-11,15/” trp1-1/” can1-100/ ”; haploid wild type = MAT a ade2-1 ura3-1 leu2-3,112 his3-11,15 trp1-1 can1-100 .aSawarynski et al (2009). …”

“…In our previous studies, we found that deletion of DMC1 blocks the DNA rereplication induced by Sic1 stabilization (Sawarynski et al 2009). In this report, we describe our further genetic investigation into constituents of the meiotic recombination checkpoint as they pertain to Sic1-induced DNA rereplication.…”

“…Cyclin-dependent kinase (CDK) complexes are central regulators of eukaryotic DNA replication initiation, both in the mitotic cell cycle (Siddiqui et al 2013) and in meiosis (Dirick et al 1998; Stuart and Wittenberg 1998; Benjamin et al 2003). We have shown in Saccharomyces cerevisiae that expression of a stabilized form of the B-type cyclin-CDK inhibitor Sic1 during meiosis can lead to extra rounds of DNA replication (Sawarynski et al 2009). This observation is consistent with the well-established role of CDK, particularly Clb5-Cdk1, in preventing DNA rereplication during the mitotic cell cycle through several mechanisms that serve to inhibit reformation of the prereplicative complex (Nguyen et al 2001; Ikui et al 2007; Siddiqui et al 2013).…”