The Mre11/Rad50/Nbs1 (MRN) complex is required for eukaryotic DNA double-strand break (DSB) repair and meiotic recombination. We cloned the Coprinus cinereus rad50 gene and showed that it corresponds to the complementation group previously named rad12, identified mutations in 15 rad50 alleles, and mapped two of the mutations onto molecular models of Rad50 structure. We found that C. cinereus rad50 and mre11 mutants arrest in meiosis and that this arrest is Spo11 dependent. In addition, some rad50 alleles form inducible, Spo11-dependent Rad51 foci and therefore must be forming meiotic DSBs. Thus, we think it likely that arrest in both mre11-1 and the collection of rad50 mutants is the result of unrepaired or improperly processed DSBs in the genome and that Rad50 and Mre11 are dispensable in C. cinereus for DSB formation, but required for appropriate DSB processing. We found that the ability of rad50 mutant strains to form Rad51 foci correlates with their ability to promote synaptonemal complex formation and with levels of stable meiotic pairing and that partial pairing, recombination initiation, and synapsis occur in the absence of wild-type Rad50 catalytic domains. Examination of single-and double-mutant strains showed that a spo11 mutation that prevents DSB formation enhances axial element (AE) formation for rad50-4, an allele predicted to encode a protein with intact hook region and hook-proximal coiled coils, but not for rad50-1, an allele predicted to encode a severely truncated protein, or for rad50-5, which encodes a protein whose hook-proximal coiled-coil region is disrupted. Therefore, Rad50 has an essential structural role in the formation of AEs, separate from the DSB-processing activity of the MRN complex. M EIOSIS is the unique type of cell division that results in the production of haploid gametes. During prophase I, homologs condense, pair, and recombine. At least one recombination event per homologous pair becomes a crossover, which, in combination with sister-chromatid cohesion, creates a chiasma that holds homologs together as they attach to the metaphase I spindle. Release of sister-chromatid cohesion along chromosome arms allows anaphase I separation of homologs, and release of centromere cohesion allows anaphase II separation of sister chromatids.Meiotic recombination events initiate with a Spo11-catalized DNA double-strand break (DSB), and some of the proteins required for meiotic recombination have been recruited from mitotic DNA repair pathways. A primary example is the complex of proteins referred to as Mre11, Rad50, and Nbs1 (MRN). Evidence from Saccharomyces cerevisiae, in which the Nbs1 ortholog is called Xrs2, showed that the MRN complex is necessary for meiotic DSB formation by Spo11 reviewed in Borde 2007). However, in other species different results have been obtained. In Caenorhabditis elegans, Rad50 facilitates DSB formation by Spo11 but is apparently not strictly required for Spo11 activity (Hayashi et al. 2007). In Schizosaccharomyces pombe (Young et al. In all eukary...

Organisms possess at least two multigene families of ubiquitins: the polyubiquitins, with few to several repeat units, which encode a ubiquitin monomer, and the ubiquitin fusion (or extension) protein genes, which encode a single ubiquitin monomer and a specific protein. This report provides details about two ubiquitin fusion protein genes in maize referred to as MubG7 (uwo 1) and MubG10 (uwo 2). Each has one nearly identical ubiquitin coding unit fused without an intervening nucleotide to an unrelated, 237-nucleotide sequence that encodes for a 79 amino acid protein. The derived amino acid sequences of the two fusion proteins show that they differ by five amino acids (substitution by either a serine or threonine). MubG7 maps to chromosome 8L162 and MubG10 maps to chromosome 1L131. Analyses of the role(s) of these genes in response to heat shock (1 h at 42.5 degrees C) reveal that the level of these fusion protein mRNAs in the radicles or plumules from 2-day-old seedlings does not change; however, heat shock does cause a marked reduction in the accumulation of these same gene-specific mRNAs in the radicles and plumules of 5-day-old seedlings. These data confirm the suggestion from our earlier work that there is precise modulation, in a gene-specific manner, of the response to developmental as well as environmental signals.

The Mre11-Rad50-Nbs1 (MRN) complex is required for numerous cellular processes that involve interactions with DNA double-strand breaks. For the majority of these processes, the MRN complex is thought to act as a unit, with each protein aiding the activity of the others. We have examined the relationship between Mre11 and Rad50 during meiosis in the basidiomycete Coprinus cinereus (Coprinopsis cinerea), investigating to what extent activities of Mre11 and Rad50 are interdependent. We showed that mre11-1 is epistatic to rad50-1 with respect to the time of meiotic arrest, indicating that Mre11 activity facilitates the diffuse diplotene arrest of rad50 mutants. Anti-Mre11 and anti-Rad50 antibodies were used to examine MRN complex localization in a wild-type strain and in spo11, mre11, and rad50 mutants. In wild type, numbers of Mre11 and Rad50 foci peaked at time points corresponding to leptotene and early zygotene. In the spo11-1 mutant, which is defective in meiotic double-strand break formation, foci accumulated throughout prophase I. Of seven MRN mutants examined, only two rad50 strains exhibited Mre11 and Rad50 foci that localized to chromatin, although Mre11 protein was found in the cell for all of them. Analysis of predicted mutant structures showed that stable localization of Mre11 and Rad50 does not depend upon a wild-type hook-proximal coiled coil, but does require the presence of the Rad50 ATPase/adenylate cyclase domains. We found that Mre11 and Rad50 were interdependent for binding to meiotic chromosomes. However, the majority of foci observed apparently contained only one of the two proteins. Independent Mre11 and Rad50 foci might indicate disassociation of the complex during meiosis or could reflect independent structural roles for the two proteins in meiotic chromatin.

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