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Meiosis is a fundamental process in eukaryotes. Homologous chromosomes are paired and recombined during meiotic prophase I, which results in variation among the gametes. However, the mechanism of recombination between the maternal and paternal chromosome is unknown. In this study, we report on the identification of interaction between Coprinus cinereus DNA polymerase mu (CcPol mu) and CcLim15/Dmc1, a meiosis-specific RecA-like protein, during meiosis. Interaction between these two proteins was confirmed using a GST-pull down assay. A two-hybrid assay revealed that the N-terminus of CcPol mu, which includes the BRCT domain, is responsible for binding the C-terminus of CcLim15. Furthermore, co-immunoprecipitation experiments indicate that these two proteins also interact in the crude extract of the meiotic cell. A significant proportion of CcPol mu and CcLim15 is shown to co-localize in nuclei from the leptotene/zygotene stage to the early pachytene stage during meiotic prophase I. Moreover, CcLim15 enhances polymerase activity of CcPol mu early in the reaction. These results suggest that CcPol mu might be recruited by CcLim15 and elongate the D-loop structure during homologous recombination in meiosis.

Mer3 is an evolutionarily conserved DNA helicase that has crucial roles in meiotic recombination and crossover formation. We have identified the MER3 homolog in Coprinus cinereus (Ccmer3) and show that it is expressed in zygotene and pachytene meiocytes. Immunostaining analysis indicated that CcMer3 was localized on chromosomes at zygotene and pachytene and CcMer3 foci were more frequent on paired than unpaired chromosomes. We generated a C. cinereus mer3 mutant (#1) and found that it showed abnormal meiosis progression and underwent apoptosis after prophase I. Basidiospore production in #1 was reduced to 0.8% of the wild-type level; the spores showed slower germination at 25 degrees C but were similar to the wild type at 37 degrees C. Electron microscopic analysis of chromosome spreads revealed that axial elements were formed in the mutant but that synapsis was defective, resulting in a reduction in spore production. Our results demonstrate that CcMer3 is required for synaptonemal complex formation after axial elements align and is thus essential for homologous synapsis.

The X family DNA polymerases lambda (CcPollambda) and mu (CcPolmu) were shown to be expressed during meiotic prophase in the basidiomycete, Coprinus cinereus. These two polymerases are the only members of the X family in the C. cinereus genome. The open reading frame of CcPollambda encoded a predicted product of 800 amino acid residues and that of CcPolmicro of 621 amino acid residues. Both CcPollambda and CcPolmicro required Mn(2+) ions for activity, and both were strongly inhibited by dideoxythymidine triphosphate. Unlike their mammalian counterparts, CcPollambda and CcPolmicro had no terminal deoxynucleotidyl transferase activity. Immunostaining analysis revealed that CcPollambda was present at meiotic prophase nuclei in zygotene and pachytene cells, which is the period when homologous chromosomes pair and recombine. CcPolmicro was present in a slightly wider range of cell stages, zygotene to diplotene. In analyses using D-loop recombination intermediate substrates, we found that both CcPollambda and CcPolmicro could promote primer extension of an invading strand in a D-loop structure. Moreover, both polymerases could fully extend the primer in the D-loop substrate, suggesting that D-loop extension is an activity intrinsic to CcPollambda and CcPolmicro. Based on these data, we discuss the possible roles of these polymerases in meiosis.

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.

Sumoylation is a post-translational modification system that covalently attaches the small ubiquitin-related modifier (SUMO) to target proteins. Ubc9 is required as the E2-type enzyme for SUMO-1 conjugation to targets. Here, we show that Ubc9 interacts with the meiosis-specific RecA homolog, Lim15/Dmc1 in the basidiomycete Coprinus cinereus (CcLim15), and mediates sumoylation of CcLim15 during meiosis. In vitro protein-protein interaction assays revealed that CcUbc9 interacts with CcLim15 and binds to the C-terminus (amino acids 105-347) of CcLim15, which includes the ATPase domain. Immunocytochemistry demonstrates that CcUbc9 and CcLim15 colocalize in the nuclei from the leptotene stage to the early pachytene stage during meiotic prophase I. Coimmunoprecipitation experiments indicate that CcUbc9 interacts with CcLim15 in vivo during meiotic prophase I. Furthermore, we show that CcLim15 is a target protein of sumoylation both in vivo and in vitro, and identify the C-terminus (amino acids 105-347) of CcLim15 as the site of sumoylation in vitro. These results suggest that sumoylation is a candidate modulator of meiotic recombination via interaction between Ubc9 and Lim15/Dmc1.

The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. The LIM15/DMC1-repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research.

Lim15/Dmc1 is a meiosis specific RecA-like protein. Here we propose its participation in meiotic chromosome pairing-related events along with DNA topoisomerase II. Analysis of protein-protein interactions using in vitro binding assays provided evidence that Coprinus cinereus DNA topoisomerase II (CcTopII) specifically interacts with C.cinereus Lim15/Dmc1 (CcLim15). Co-immunoprecipitation experiments also indicated that the CcLim15 protein interacts with CcTopII in vivo. Furthermore, a significant proportion of CcLim15 and CcTopII could be shown to co-localize on chromosomes from the leptotene to the zygotene stage. Interestingly, CcLim15 can potently activate the relaxation/catenation activity of CcTopII in vitro, and CcTopII suppresses CcLim15-dependent strand transfer activity. On the other hand, while enhancement of CcLim15's DNA-dependent ATPase activity by CcTopII was found in vitro, the same enzyme activity of CcTopII was inhibited by adding CcLim15. The interaction of CcLim15 and CcTopII may facilitate pairing of homologous chromosomes.