Interplay netween Dbf4-dependent Cdc7 kinase and polo-like kinase unshackles mitotic recombination mechanisms by promoting synaptonemal complex disassembly

Meiotic recombination is initiated by self-inflicted DNA breaks and primarily involves homologous chromosomes, whereas mitotic recombination involves sister chromatids. Whilst the mitotic recombinase Rad51 exists during meiosis, its activity is suppressed in favour of the meiosis-specific recombinase, Dmc1, thus establishing a meiosis-specific mode of homologous recombination (HR). A key contributor to the suppression of Rad51 activity is the synaptonemal complex (SC), a meiosis-specific chromosomal structure that adheres homologous chromosomes along their entire lengths. Here, in budding yeast, we show that two major cell cycle kinases, Dbf4-dependent Cdc7 kinase (DDK) and Polo-kinase (Cdc5), collaborate to link the mode change of HR to the meiotic cell cycle by. This regulation of HR is through the SC. During prophase I, DDK is shown to maintain SC integrity and thus inhibition of Rad51. Cdc5, which is produced during the prophase I/metaphase I transition, interacts with DDK to cooperatively destroy the SC and remove Rad51 inhibition. By enhancing the interaction between DDK and Cdc5 or depleting DDK at late prophase I, meiotic DNA breaks are repaired even in the absence of Dmc1 by utilising Rad51. We propose that the interplay between DDK and Polo-kinase reactivates mitotic HR mechanisms to ensure complete repair of DNA breaks before meiotic chromosomem segregation

Direct and indirect control of the initiation of meiotic recombination by DNA damage checkpoint mechanisms in budding yeast

Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs). The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM) pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR) pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI) whereas no significant reduction was found in smaller chromosomes (III and VI). On the other hand, the absence of Rad17 (a critical component of the ATR pathway) lead to an increase in DSB formation (chromosomes VII and II were tested). We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation

The Ecm11-Gmc2 complex promotes synaptonemal complex formation through assembly of transverse filaments in budding yeast

During meiosis, homologous chromosomes pair at close proximity to form the synaptonemal complex (SC). This association is mediated by transverse filament proteins that hold the axes of homologous chromosomes together along their entire length. Transverse filament proteins are highly aggregative and can form an aberrant aggregate called the polycomplex that is unassociated with chromosomes. Here, we show that the Ecm11-Gmc2 complex is a novel SC component, functioning to facilitate assembly of the yeast transverse filament protein, Zip1. Ecm11 and Gmc2 initially localize to the synapsis initiation sites, then throughout the synapsed regions of paired homologous chromosomes. The absence of either Ecm11 or Gmc2 substantially compromises the chromosomal assembly of Zip1 as well as polycomplex formation, indicating that the complex is required for extensive Zip1 polymerization. We also show that Ecm11 is SUMOylated in a Gmc2-dependent manner. Remarkably, in the unSUMOylatable ecm11 mutant, assembly of chromosomal Zip1 remained compromised while polycomplex formation became frequent. We propose that the Ecm11-Gmc2 complex facilitates the assembly of Zip1 and that SUMOylation of Ecm11 is critical for ensuring chromosomal assembly of Zip1, thus suppressing polycomplex formation

Budding Yeast Pch2, a Widely Conserved Meiotic Protein, Is Involved in the Initiation of Meiotic Recombination

Budding yeast Pch2 protein is a widely conserved meiosis-specific protein whose role is implicated in the control of formation and displacement of meiotic crossover events. In contrast to previous studies where the function of Pch2 was implicated in the steps after meiotic double-strand breaks (DSBs) are formed, we present evidence that Pch2 is involved in meiotic DSB formation, the initiation step of meiotic recombination. The reduction of DSB formation caused by the pch2 mutation is most prominent in the sae2 mutant background, whereas the impact remains mild in the rad51 dmc1 double mutant background. The DSB reduction is further pronounced when pch2 is combined with a hypomorphic allele of SPO11. Interestingly, the level of DSB reduction is highly variable between chromosomes, with minimal impact on small chromosomes VI and III. We propose a model in which Pch2 ensures efficient formation of meiotic DSBs which is necessary for igniting the subsequent meiotic checkpoint responses that lead to proper differentiation of meiotic recombinants

Shaping meiotic chromosomes with SUMO: a feedback loop controls the assembly of the synaptonemal complex in budding yeast

The synaptonemal complex (SC) is a meiosis-specific chromosomal structure in which homologous chromosomes are intimately linked through arrays of specialized proteins called transverse filaments (TF). Widely conserved in eukaryote meiosis, the SC forms during prophase I and is essential for accurate segregation of homologous chromosomes at meiosis I. However, the basic mechanism overlooking formation and regulation of the SC has been poorly understood. By using the budding yeast Saccharomyces cerevisiae, we recently showed that SC formation is controlled through the attachment of multiple molecules of small ubiquitin-like modifier (SUMO) to a regulator of TF assembly. Intriguingly, this SUMOylation is activated by TF, implicating the involvement of a positive feedback loop in the control of SC assembly. We discuss the implication of this finding and possible involvement of a similar mechanism in regulating other processes

Ecm11 and Gmc2 are necessary for the efficient assembly of Zip1 on chromosomes and in the polycomplex.

<p>(A) Zip1 localization is discontinuous on meiotic prophase chromosomes in the <i>ecm11</i> and <i>gmc2</i> mutants. The localization of Zip1 along with Red1, a component of meiotic chromosome axes, was examined on spread chromosomes. Bar, 5 µm. (B, C) Quantitative analysis of the Zip1 localization. Zip1 stretch area represents the size of one continuous Zip1 staining. See Results and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003194#s4" target="_blank">Materials and Methods</a> for more details. (D) Polycomplex formation was abolished in <i>ecm11</i> and <i>gmc2</i> mutants. Zip1 localization was examined in the <i>spo11</i> mutant background. The white arrowhead indicates the location of the polycomplex. Small speckle-like Zip1 staining likely represents the centromeric localization of Zip1. Chromosome spreads were prepared using cells at 20 hours after introduction into meiosis when, in wild type, cells at the pachytene stage are enriched. (E) Quantification of spread nuclei exhibiting a polycomplex.</p

SUMOylation of Ecm11 at Lysine 5 is essential for facilitating the chromosomal assembly of Zip1.

<p>(A) The predominant role of Lysine 5 in facilitating the chromosomal assembly of Zip1. Meiotic chromosomes of wild type cells or <i>ecm11</i> mutants in which SUMOylation was compromised were examined for their Zip1 localization and also for Red1. Bar, 5 µm. (B, C) Quantitative analyses of Zip1 localization. See Figure legend of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003194#pgen-1003194-g002" target="_blank">Figure 2B, 2C</a> and also Results and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003194#s4" target="_blank">Materials and Methods</a> for details. (D) Polycomplex formation was elevated when Lysine 5 was mutated. Meiotic chromosomes carrying the mutations indicated were stained for the Zip1 protein and the fractions of chromosome spreads carrying a polycomplex were calculated. At least 100 chromosome spreads were counted for each strain.</p