Crossing over establishes connections between homologous chromosomes that promote their proper segregation at the first meiotic division. However, there exists a backup system to ensure the correct segregation of those chromosome pairs that fail to cross over. We have found that, in budding yeast, a mutation eliminating the synaptonemal complex protein, Zip1, increases the meiosis I nondisjunction rate of nonexchange chromosomes (NECs). The centromeres of NECs become tethered during meiotic prophase, and this tethering is disrupted by the zip1 mutation. Furthermore, the Zip1 protein often colocalizes to the centromeres of the tethered chromosomes, suggesting that Zip1 plays a direct role in holding NECs together. Zip3, a protein involved in the initiation of synaptonemal complex formation, is also important for NEC segregation. In the absence of Zip3, both the tethering of NECs and the localization of Zip1 to centromeres are impaired. A mutation in the MAD3 gene, which encodes a component of the spindle checkpoint, also increases the nondisjunction of NECs. Together, the zip1 and mad3 mutations have an additive effect, suggesting that these proteins act in parallel pathways to promote NEC segregation. We propose that Mad3 promotes the segregation of NECs that are not tethered by Zip1 at their centromeres.

Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin complexes. Furthermore, our data suggests that STAG3 is required for structural changes of chromosomes that mediate chromosome pairing and synapsis, DNA repair and progression of meiosis.

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4Eme1. Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastrophe.

During meiosis, accurate coordination of the completion of homologous recombination and synaptonemal complex (SC) disassembly during the prophase to metaphase I (G2/MI) transition is essential to avoid aneuploid gametes and infertility. Previous studies have shown that kinase activity is required to promote meiotic prophase exit. The first step of the G2/MI transition is the disassembly of the central element components of the SC; however, the kinase(s) required to trigger this process remains unknown. Here we assess roles of polo-like kinases (PLKs) in mouse spermatocytes, both in vivo and during prophase exit induced ex vivo by the phosphatase inhibitor okadaic acid. All four PLKs are expressed during the first wave of spermatogenesis. Only PLK1 (not PLK2-4) localizes to the SC during the G2/MI transition. The SC central element proteins SYCP1, TEX12 and SYCE1 are phosphorylated during the G2/MI transition. However, treatment of pachytene spermatocytes with the PLK inhibitor BI 2536 prevented the okadaic-acid-induced meiotic prophase exit and inhibited phosphorylation of the central element proteins as well as their removal from the SC. Phosphorylation assays in vitro demonstrated that PLK1, but not PLK2-4, phosphorylates central element proteins SYCP1 and TEX12. These findings provide mechanistic details of the first stage of SC disassembly in mammalian spermatocytes, and reveal that PLK-mediated phosphorylation of central element proteins is required for meiotic prophase exit.

SummaryFour members of the structural maintenance of chromosome (SMC) protein family have essential functions in chromosome condensation (SMC2/4) and sister-chromatid cohesion (SMC1/3). The SMC5/6 complex has been implicated in chromosome replication, DNA repair and chromosome segregation in somatic cells, but its possible functions during mammalian meiosis are unknown. Here, we show in mouse spermatocytes that SMC5 and SMC6 are located at the central region of the synaptonemal complex from zygotene until diplotene. During late diplotene both proteins load to the chromocenters, where they colocalize with DNA Topoisomerase IIa, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase IIa colocalize at stretched strands that join kinetochores during the metaphase II to anaphase II transition, and both are observed on stretched lagging chromosomes at anaphase II following treatment with Etoposide. During mitosis, SMC6 and DNA Topoisomerase IIa colocalize at the centromeres and chromatid axes. Our results are consistent with the participation of SMC5 and SMC6 in homologous chromosome synapsis during prophase I, chromosome and centromere structure during meiosis I and mitosis and, with DNA Topoisomerase IIa, in regulating centromere cohesion during meiosis II.

Mutations in centrosome genes deplete neural progenitor cells (NPCs) during brain development, causing microcephaly. While NPC attrition is linked to TP53-mediated cell death in several microcephaly models, how TP53 is activated remains unclear. In cultured cells, mitotic delays resulting from centrosome loss prevent the growth of unfit daughter cells by activating a pathway involving 53BP1, USP28, and TP53, termed the mitotic surveillance pathway. Whether this pathway is active in the developing brain is unknown. Here, we show that the depletion of centrosome proteins in NPCs prolongs mitosis and increases TP53-mediated apoptosis. Cell death after a delayed mitosis was rescued by inactivation of the mitotic surveillance pathway. Moreover, 53BP1 or USP28 deletion restored NPC proliferation and brain size without correcting the upstream centrosome defects or extended mitosis. By contrast, microcephaly caused by the loss of the non-centrosomal protein SMC5 is also TP53-dependent but is not rescued by loss of 53BP1 or USP28. Thus, we propose that mutations in centrosome genes cause microcephaly by delaying mitosis and pathologically activating the mitotic surveillance pathway in the developing brain.

Several protein kinases collaborate to orchestrate and integrate cellular and chromosomal events at the G2/M transition in both mitotic and meiotic cells. During the G2/M transition in meiosis, this includes the completion of crossover recombination, spindle formation, and synaptonemal complex (SC) breakdown. We identified Ipl1/ Aurora B kinase as the main regulator of SC disassembly. Mutants lacking Ipl1 or its kinase activity assemble SCs with normal timing, but fail to dissociate the central element component Zip1, as well as its binding partner, Smt3/SUMO, from chromosomes in a timely fashion. Moreover, lack of Ipl1 activity causes delayed SC disassembly in a cdc5 as well as a CDC5-inducible ndt80 mutant. Crossover levels in the ipl1 mutant are similar to those observed in wild type, indicating that full SC disassembly is not a prerequisite for joint molecule resolution and subsequent crossover formation. Moreover, expression of meiosis I and meiosis II-specific B-type cyclins occur normally in ipl1 mutants, despite delayed formation of anaphase I spindles. These observations suggest that Ipl1 coordinates changes to meiotic chromosome structure with resolution of crossovers and cell cycle progression at the end of meiotic prophase.

Neisseria meningitidis, a causative agent of bacterial meningitis, has a relatively small repertoire of transcription factors, including NMB0573 (annotated AsnC), a member of the LrpAsnC family of regulators that are widely expressed in both Bacteria and Archaea. In the present study we show that NMB0573 binds to L-leucine and L-methionine and have solved the structure of the protein with and without bound amino acids. This has shown, for the first time that amino acid binding does not induce significant conformational changes in the structure of an AsnC/Lrp regulator although it does appear to stabilize the octameric assembly of the protein. Transcriptional profiling of wild-type and NMB0573 knock-out strains of N. meningitidis has shown that NMB0573 is associated with an adaptive response to nutrient poor conditions reflected in a reduction in major surface protein expression. On the basis of its structure and the transcriptional response, we propose that NMB0573 is a global regulator in Neisseria controlling responses to nutrient availability through indicators of general amino acid abundance: leucine and methionine.The L-leucine-responsive regulatory protein (Lrp) 2 /AsnC family of transcription factors are widely distributed in both Bacteria and Archaea but do not appear to be present in eukaryotic genomes. The family is named after two proteins from Escherichia coli implicated in the control of amino acid metabolism; the Lrp and AsnC, which share ϳ25% sequence identity. Lrp is a global regulator that controls the expression of a large number of operons in E. coli, including those involved in the synthesis and degradation of amino acids (1), whereas AsnC is a specific regulator of the asnA gene, which codes for asparagine synthetase, responsible for converting aspartate to asparagine in an ATP-dependent reaction, and an autorepressor of its own expression (2). As the name implies, many Lrp-responsive operons are co-regulated by L-leucine that can either antagonize or potentiate the effects of Lrp (3). L-Leucine is one of the most common amino acids in proteins and the reciprocal effect of Lrp on amino acid metabolism suggests that intracellular Lrp abundance mediates transitions between "feast and famine" (3, 4). AsnC, on the other hand, appears to be responsible for controlling asparagine levels via negative feedback regulation of asparagine synthetase expression (2, 5).Insight into the relationship between the structure of Lrp/ AsnC family proteins and their function has come from x-ray crystallography. To date, four x-ray crystal structures from the family have been reported, two from Archaeal sources; Pyrococcus furiosus LrpA (Protein Data Bank code 1I1G (6) and the close homologue, FL11, from Pyrococcus sp. OT3 (PDB code 1RI7 (7) and two from bacterial sources: E. coli AsnC in complex with asparagine (PDB code 2CG4) and Bacillus subtilis LrpC (PDB code 2CFX) (8). The Lrp/AsnC family fold revealed by analysis of these structures consists of a N-terminal DNAbinding domain containing a helix-turn-helix ...