The synaptonemal complex is assembled by a polySUMOylation-driven feedback mechanism in yeast.

During meiotic prophase I, proteinaceous structures called synaptonemal complexes (SCs) connect homologous chromosomes along their lengths via polymeric arrays of transverse filaments (TFs). Thus, control of TF polymerization is central to SC formation. Using budding yeast, we show that efficiency of TF polymerization closely correlates with the extent of SUMO conjugation to Ecm11, a component of SCs. HyperSUMOylation of Ecm11 leads to highly aggregative TFs, causing frequent assembly of extrachromosomal structures. In contrast, hypoSUMOylation leads to discontinuous, fragmented SCs, indicative of defective TF polymerization. We further show that the N terminus of the yeast TF, Zip1, serves as an activator for Ecm11 SUMOylation. Coexpression of the Zip1 N terminus and Gmc2, a binding partner of Ecm11, is sufficient to induce robust polySUMOylation of Ecm11 in nonmeiotic cells. Because TF assembly is mediated through N-terminal head-to-head associations, our results suggest that mutual activation between TF assembly and Ecm11 polySUMOylation acts as a positive feedback loop that underpins SC assembly.

UV induced ubiquitination of the yeast Rad4-Rad23 complex promotes survival by regulating cellular dNTP pools.

Regulating gene expression programmes is a central facet of the DNA damage response. The Dun1 kinase protein controls expression of many DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Using a combination of gene expression profiling and chromatin immunoprecipitation, we demonstrate that in the absence of DNA damage the yeast Rad4-Rad23 nucleotide excision repair complex binds to the promoters of certain DNA damage response genes including DUN1, inhibiting their expression. UV radiation promotes the loss of occupancy of the Rad4-Rad23 complex from the regulatory regions of these genes, enabling their induction and thereby controlling the production of dNTPs. We demonstrate that this regulatory mechanism, which is dependent on the ubiquitination of Rad4 by the GG-NER E3 ligase, promotes UV survival in yeast cells. These results support an unanticipated regulatory mechanism that integrates ubiquitination of NER DNA repair factors with the regulation of the transcriptional response controlling dNTP production and cellular survival after UV damage.

A non-sister act: recombination template choice during meiosis.

Meiotic recombination has two key functions: the faithful assortment of chromosomes into gametes and the creation of genetic diversity. Both processes require that meiotic recombination occurs between homologous chromosomes, rather than sister chromatids. Accordingly, a host of regulatory factors are activated during meiosis to distinguish sisters from homologs, suppress recombination between sister chromatids and promote the chromatids of the homologous chromosome as the preferred recombination partners. Here, we discuss the recent advances in our understanding of the mechanistic basis of meiotic recombination template choice, focusing primarily on developments in the budding yeast, Saccharomyces cerevisiae, where the regulation is currently best understood.

SUMO localizes to the central element of synaptonemal complex and is required for the full synapsis of meiotic chromosomes in budding yeast.

The synaptonemal complex (SC) is a widely conserved structure that mediates the intimate alignment of homologous chromosomes during meiotic prophase and is required for proper homolog segregation at meiosis I. However, fundamental details of SC architecture and assembly remain poorly understood. The coiled-coil protein, Zip1, is the only component whose arrangement within the mature SC of budding yeast has been extensively characterized. It has been proposed that the Small Ubiquitin-like MOdifier, SUMO, plays a role in SC assembly by linking chromosome axes with Zip1's C termini. The role of SUMO in SC structure has not been directly tested, however, because cells lacking SUMO are inviable. Here, we provide direct evidence for SUMO's function in SC assembly. A meiotic smt3 reduction-of-function strain displays reduced sporulation, abnormal levels of crossover recombination, and diminished SC assembly. SC structures are nearly absent when induced at later meiotic time points in the smt3 reduction-of-function background. Using Structured Illumination Microscopy we furthermore determine the position of SUMO within budding yeast SC structure. In contrast to previous models that positioned SUMO near Zip1's C termini, we demonstrate that SUMO lies at the midline of SC central region proximal to Zip1's N termini, within a subdomain called the "central element". The recently identified SUMOylated SC component, Ecm11, also localizes to the SC central element. Finally, we show that SUMO, Ecm11, and even unSUMOylatable Ecm11 exhibit Zip1-like ongoing incorporation into previously established SCs during meiotic prophase and that the relative abundance of SUMO and Ecm11 correlates with Zip1's abundance within SCs of varying Zip1 content. We discuss a model in which central element proteins are core building blocks that stabilize the architecture of SC near Zip1's N termini, and where SUMOylation may occur subsequent to the incorporation of components like Ecm11 into an SC precursor structure.

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.