Repair of double-strand DNA breaks (DSBs) by the homologous recombination (HR) pathway results in crossovers (COs) required for a successful first meiotic division. Mre11 is one member of the MRX/N (Mre11, Rad50, and Xrs2/Nbs1) complex required for meiotic DSB formation and for resection in Saccharomyces cerevisiae. In Caenorhabditis elegans, evidence for the MRX/N role in DSB resection is limited. We report the first separation-of-function allele, mre-11(iow1) in C. elegans, which is specifically defective in meiotic DSB resection but not in formation. The mre-11(iow1) mutants displayed chromosomal fragmentation and aggregation in late prophase I. Recombination intermediates and crossover formation was greatly reduced in mre-11(iow1) mutants. Irradiation-induced DSBs during meiosis failed to be repaired from early to middle prophase I in mre-11(iow1) mutants. In the absence of a functional HR, our data suggest that some DSBs in mre-11(iow1) mutants are repaired by the nonhomologous end joining (NHEJ) pathway, as removing NHEJ partially suppressed the meiotic defects shown by mre-11(iow1). In the absence of NHEJ and a functional MRX/N, meiotic DSBs are channeled to EXO-1-dependent HR repair. Overall, our analysis supports a role for MRE-11 in the resection of DSBs in middle meiotic prophase I and in blocking NHEJ. M eiotic recombination is initiated by the formation of meiotic double-strand breaks (DSBs), which are then repaired by the meiotic homologous recombination (HR) pathway. A meiosis protein, SPO-11, along with other auxiliary proteins catalyzes the formation of meiotic DSBs and remains covalently bound to the 5= ends of the DSBs (1). These DSBs are converted to long 3= singlestranded DNA (ssDNA) by a process called resection. Meiotic DSB resection is initiated by endonuclease activity to release oligonucleotide-bound SPO-11 and generate a short 3= overhang (2). The short 3= overhang is further processed by a 5=-3= exonuclease activity to generate long 3= ssDNA (3, 4). The ssDNA is rapidly bound by the ssDNA binding protein RPA, which is then displaced by RAD-51, forming a filament that coats the ssDNA (5). RAD-51 then mediates the invasion of the ssDNA into the homologous double-strand DNA (dsDNA) (6, 7). Using the homologous sequence as the template, new DNA is synthesized from the invading 3= ends to fill in the region lost by end resection.

Formation of a condensed and properly remodeled bivalent is required for accurate execution of meiosis. Meiotic roles are identified for the highly evolutionarily conserved protein AKIRIN in bivalent remodeling in a synaptonemal complex (SC)–dependent and SC–independent manner, demonstrating that proper SC disassembly is crucial for bivalent structure.

The synaptonemal complex (SC) is a conserved protein structure that holds homologous chromosome pairs together throughout much of meiotic prophase I. It is essential for the formation of crossovers, which are required for the proper segregation of chromosomes into gametes. The assembly of the SC is likely to be regulated by post-translational modifications. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via the ubiquitin degradation/proteasome pathway. Here we examine the role of the CSN/COP9 signalosome in SC assembly in the model organism C. elegans. Our work shows that mutants in three subunits of the CSN/COP9 signalosome fail to properly assemble the SC. In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes. The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers. The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes. We also find a marked increase in apoptosis in csn mutants that specifically eliminates nuclei with aggregated SC proteins. csn mutants exhibit defects in germline proliferation, and an almost complete pachytene arrest due to an inability to activate the MAPK pathway. The work described here supports a previously unknown role for the CSN/COP9 signalosome in chromosome behavior during meiotic prophase I.

Meiosis is a specialized cellular division occurring in organisms capable of sexual reproduction that leads to the formation of gametes containing half of the original chromosome number. During the earliest stage of meiosis, prophase I, pairing of homologous chromosomes is achieved in preparation for their proper distribution in the coming divisions. An important question is how do homologous chromosomes find each other and establish pairing interactions. Early studies demonstrated that chromosomes are dynamic in nature and move during this early stage of meiosis. More recently, there have been several studies across different models showing the conserved nature and importance of this chromosome movement, as well as the key components involved in chromosome movement. This review will cover these major findings and also introduce unexamined areas of regulation in meiotic prophase I chromosome movement.

During meiotic prophase I, sister chromatid cohesion is established in a way that supports the assembly of the synaptonemal complex (SC). The SC connects homologous chromosomes, directing meiotic recombination to create crossovers. In this paper, we identify two proteins that cooperate to import and load meiotic cohesins, thus indirectly promoting SC assembly. AKIR-1 is a protein with a previously identified meiotic role in SC disassembly. akir-1 mutants have no obvious defects in sister chromatid cohesion. We identified ima-2, a gene encoding for an a-importin nuclear transport protein, as a gene interacting with akir-1. Analysis of akir-1;ima-2 double mutants reveals a decrease in the number of germline nuclei and the formation of polycomplexes (PCs) (an SC protein aggregate). These PCs contain proteins that are part of the two main substructures of the SC: the central region and the lateral element. Unlike typical PCs, they also contain sister chromatid cohesion proteins. In akir-1;ima-2 double mutants, PCs are located in both the nucleus and the cytoplasm. This suggests that the defects observed in the double mutants are both in nuclear import and in the assembly of sister chromatid cohesion. PC formation is also associated with recombination defects leading to reduced numbers of crossovers. Similarly to cohesion mutants, the pairing center protein HIM-8 is mislocalized in akir-1;ima-2 double mutants, forming multiple foci. We propose that AKIR-1 and IMA-2 operate in parallel pathways to import and load chromosomally associated cohesin complex proteins in meiotic nuclei, a novel finding for both of these conserved proteins.

Identifying protein localization is a useful tool in analyzing protein function. Using GFP-fusion tags, researchers can study the function of endogenous proteins in living tissue. However, these tags are considerably large, making them difficult to insert, and they can potentially affect the normal function of these proteins. To improve on these drawbacks, we have adopted the split sfGFP system for studying the localization of proteins in the Caenorhabditis elegans germline. This system divides the “super folder” GFP into 2 fragments, allowing researchers to use CRISPR/Cas9 to tag proteins more easily with the smaller subunit, while constitutively expressing the larger subunit from another locus. These two parts are able to stably interact, producing a functional GFP when both fragments are in the same cellular compartment. Our data demonstrate that the split sfGFP system can be adapted for use in C. elegans to tag endogenous proteins with relative ease. Strains containing the tags are homozygous viable and fertile. These small subunit tags produce fluorescent signals that matched the localization patterns of the wild-type protein in the gonad. Thus, our study shows that this approach could be used for tissue-specific GFP expression from an endogenous locus.

Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence.

Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase's role in germline development in higher eukaryotes.KEYWORDS KIN-18; MPK-1; TAO; meiosis; recombination; genetics of sex T HE germline contains a multitude of cells that proliferate to form a population that will undergo two meiotic divisions, giving rise to gametes. In meiosis, one round of DNA replication is followed by two cell divisions reducing the ploidy of the germ cell by half. A large fraction of the cells encompassing the germline are found in meiotic prophase I. Many of the key evolutionarily conserved events that take place in this specialized prophase are aimed at preparing chromosomes for their segregation in meiosis I. One such essential and conserved event is the formation of crossovers between homologous chromosomes. During the initial stages of meiotic prophase I (leptotene/zygotene), a protein structure named the synaptonemal complex (SC) starts to assemble between homologs (MacQueen et al. 2002;Schild-Prufert et al. 2011). Concurrently, meiotic DNA double-strand breaks (DSBs) are introduced to initiate meiotic recombination (Keeney et al. 1997;Dernburg et al. 1998). As meiosis progresses, germ cells enter pachytene where the SC is fully formed and meiotic DSBs are processed and repaired. In the presence of a fully formed SC, meiotic DSBs are repaired, giving rise to crossovers by late pachytene. In most multicellular organisms, apoptosis is activated in late pachytene and reduces the number of germ cells in the developing germline (Gumienny et al. 1999). Apoptosis serves as a quality control...