Examination of the Intron in the meiosis-specific recombination gene REC114 in Saccharomyces

REC114 is one of 10 genes known to be required for the initiation of meiotic recombination in Saccharomyces cerevisiae. It is transcribed only in meiosis, and our previous sequence analysis suggested the presence of an intron in the 3′ end of the gene. Hypotheses in the literature have suggested, because of its unusual location, either that the putative intron in REC114 is likely to be necessary for expression, or that there may actually be no intron present. This work demonstrates that REC114 does have an intron and is one of only three genes in yeast with introns located in the 3′ end. Furthermore, the 3′ splice site utilized in REC114 is a very rare AAG sequence; only three other genes in yeast use this nonconsensus sequence. The splicing of REC114 does not require MER1, a gene known to be involved in meiosis-specific RNA processing. In fact, an intronless copy of REC114 can complement a null rec114 mutation. Thus, it does not appear that the intron is essential for expression of REC114. Although the intron is not absolutely required for meiotic function, it is conserved in evolution; two other species of yeast contain an intron at the same location in their REC114 genes. Received: 16 October 1996 / Accepted: 10 February 1997     

Molecular and Genetic Analysis of Rec103, an Early Meiotic Recombination Gene in Yeast

In the yeast Saccharomyces cerevisiae at least 10 genes are required to begin meiotic recombination. A new early recombination gene REC103 is described in this paper. It was initially defined by the rec103-1 mutation found in a selection for mutations overcoming the spore inviability of a rad52 spo13 haploid strain. Mutations in REC103 also rescue rad52 in spo13 diploids. rec103 spo13 strains produce viable spores; these spores show no evidence of meiotic recombination. rec103 SPO13 diploids produce no viable spores, consistent with the loss of recombination. Mutations in REC103 do not affect mitotic recombination, growth, or repair. These phenotypes are identical to those conferred by mutations in several other early meiotic recombination genes (e.g., REC102, REC104, REC114, MEI4, MER2, and SPO11). REC103 maps to chromosome VII between ADE5 and RAD54. Cloning and sequencing of REC103 reveals that REC103 is identical to SKI8, a gene that depresses the expression of yeast double-stranded (``killer') (ds)RNA viruses. REC103/SKI8 is transcribed in mitotic cells and is induced ~15-fold in meiosis. REC103 has 26% amino acid identity to the Schizosaccharomyces pombe rec14(+) gene; mutations in both genes confer similar meiotic phenotypes, suggesting that they may play similar roles in meiotic recombination.     

The meiotic cohesin subunit REC8 contributes to multigenic adaptive evolution of autopolyploid meiosis in Arabidopsis arenosa

Genome duplication, which leads to polyploidy, poses challenges to the meiotic segregation of the now-multiple homologous chromosome copies. Genome scan data showed previously that adaptation to polyploid meiosis in autotetraploid Arabidopsis arenosa is likely multigenic, involving genes encoding interacting proteins. But what does this really mean? Functional follow-up studies to genome scans for multigenic traits remain rare in most systems, and thus many mysteries remain about the “functional architecture” of polygenic adaptations. Do different genes all contribute subtle and additive progression towards a fitness optimum, or are there more complex interactions? We previously showed that derived alleles of genes encoding two interacting meiotic axis proteins (ASY1 and ASY3) have additive functional consequences for meiotic adaptation. Here we study derived versus ancestral alleles of the meiotic cohesin subunit REC8, which has roles in chromatin condensation, recruiting the axes, and other critical functions in meiosis. We use genetic and cytological approaches to assess the functional effects of REC8 diploid versus tetraploid alleles, as well as their interaction with ancestral versus derived alleles of ASY1 and ASY3. We show that homozygotes for derived (tetraploid) REC8 alleles have significantly fewer unpaired univalents, a common problem in neotetraploids. Interactions with ASY1 and ASY3 are complex, with the genes in some cases affecting distinct traits, and additive or even antagonistic effects on others. These findings suggest that the road to meiotic adaptation in A. arenosa was perhaps neither straight nor smooth.     

Exo1 and Mre11 execute meiotic DSB end resection in the protist Tetrahymena

The resection of 5′-DNA ends at a double-strand break (DSB) is an essential step in recombinational repair, as it exposes 3′ single-stranded DNA (ssDNA) tails for interaction with a repair template. In mitosis, Exo1 and Sgs1 have a conserved function in the formation of long ssDNA tails, whereas this step in the processing of programmed meiotic DSBs is less well-characterized across model organisms. In budding yeast, which has been most intensely studied in this respect, Exo1 is a major meiotic nuclease. In addition, it exerts a nuclease-independent function later in meiosis in the conversion of DNA joint molecules into ZMM-dependent crossovers. In order to gain insight into the diverse meiotic roles of Exo1, we investigated the effect of Exo1 deletion in the ciliated protist Tetrahymena. We found that Exo1 together with Mre11, but without the help of Sgs1, promotes meiotic DSB end resection. Resection is completely eliminated only if both Mre11 and Exo1 are missing. This is consistent with the yeast model where Mre11 promotes resection in the 3′–5′ direction and Exo1 in the opposite 5′–3′ direction. However, while the endonuclease activity of Mre11 is essential to create an entry site for exonucleases and hence to start resection in budding yeast, Tetrahymena Exo1 is able to create single-stranded DNA in the absence of Mre11. Excluding a possible contribution of the Mre11 cofactor Sae2 (Com1) as an autonomous endonuclease, we conclude that there exists another unknown nuclease that initiates DSB processing in Tetrahymena. Consistent with the absence of the ZMM crossover pathway in Tetrahymena, crossover formation is independent of Exo1.     

Identification of essential intron sequences that enhance gene expression independently of splicing in the yeast Saccharomyces cerevisiae

Gene expression in eukaryotes is enhanced by the presence of introns in a process known as intron-mediated enhancement (IME), but its mechanism remains unclear. In Saccharomyces cerevisiae, sequences at the 5′-splice sites (SS) and branch point sites (BPS) are highly conserved compared with other higher eukaryotes. Here, the minimum intron sequence essential for IME was investigated using various short introns and a yeast codon-optimized luciferase gene as an IME model. Mutations at the 5′-SS conserved sequence and branch point in the QCR10 intron caused splicing deficiency with either a complete loss or a marked decrease in IME. By contrast, however, the 3′-AG to tG mutant was spliced and retained IME function. Moreover, heterologous introns, which did not show IME in S. cerevisiae, gained splicing competency and IME ability by substitutions to the S. cerevisiae-type 5′-SS and BPS sequences. Intriguingly, several deletion mutants between the 5′-SS and BPS in introns exhibited high levels of IME despite a loss in splicing competency. In most cases, further deletions or substitutions did not recover splicing competency and were found to decrease IME. However, a 16-nt variant consisting of the conserved 5′-SS and BPS sequences and 3′-CAG showed an IME level comparable with that of the wild-type intron. These results indicate that IME can be independent of splicing in S. cerevisiae while intron sequences at the 5′-SS and BPS play an essential role in IME.     

Isolation of Mutants Defective in Early Steps of Meiotic Recombination in the Yeast Saccharomyces Cerevisiae

Using a selection based upon the ability of early Rec- mutations (e.g., rad50) to rescue the meiotic lethality of a rad52 spo13 strain, we have isolated 177 mutants. Analysis of 56 of these has generated alleles of the known Rec genes SPO11, ME14 and MER1, as well as defining five new genes: REC102, REC104, REC107, REC113 and REC114. Mutations in all of the new genes appear to specifically affect meiosis; they do not have any detectable mitotic phenotype. Mutations in REC102, REC104 and REC107 reduce meiotic recombination several hundred fold. No alleles of RED1 or HOP1 were isolated, consistent with the proposal that these genes may be primarily involved with chromosome pairing and not exchange.     

Yeast Assay Highlights the Intrinsic Genomic Instability of Human PML Intron 6 over Intron 3 and the Role of Replication Fork Proteins

Human acute promyelocytic leukemia (APL) is characterized by a specific balanced translocation t(15;17)(q22;q21) involving the PML and RARA genes. In both de novo and therapy-related APL, the most frequent PML breakpoints are located within intron 6, and less frequently in intron 3; the precise mechanisms by which these breakpoints arise and preferentially in PML intron 6 remain unsolved. To investigate the intrinsic properties of the PML intron sequences in vivo, we designed Saccharomyces cerevisiae strains containing human PML intron 6 or intron 3 sequences inserted in yeast chromosome V and measured gross chromosomal rearrangements (GCR). This approach provided evidence that intron 6 had a superior instability over intron 3 due to an intrinsic property of the sequence and identified the 3’ end of intron 6 as the most susceptible to break. Using yeast strains invalidated for genes that control DNA replication, we show that this differential instability depended at least upon Rrm3, a DNA helicase, and Mrc1, the human claspin homolog. GCR induction by hydrogen peroxide, a general genotoxic agent, was also dependent on genetic context. We conclude that: 1) this yeast system provides an alternative approach to study in detail the properties of human sequences in a genetically controlled situation and 2) the different susceptibility to produce DNA breaks in intron 6 versus intron 3 of the human PML gene is likely due to an intrinsic property of the sequence and is under replication fork genetic control.     

Analysis of Meiotic Recombination Pathways in the Yeast Saccharomyces Cerevisiae

In the yeast, Saccharomyces cerevisiae, several genes appear to act early in meiotic recombination. HOP1 and RED1 have been classified as such early genes. The data in this paper demonstrate that neither a red1 nor a hop1 mutation can rescue the inviable spores produced by a rad52 spo13 strain; this phenotype helps to distinguish these two genes from other early meiotic recombination genes such as SPO11, REC104, or MEI4. In contrast, either a red1 or a hop1 mutation can rescue a rad50S spo13 strain; this phenotype is similar to that conferred by mutations in the other early recombination genes (e.g., REC104). These two different results can be explained because the data presented here indicate that a rad50S mutation does not diminish meiotic intrachromosomal recombination, similar to the mutant phenotypes conferred by red1 or hop1. Of course, RED1 and HOP1 do act in the normal meiotic interchromosomal recombination pathway; they reduce interchromosomal recombination to ~10% of normal levels. We demonstrate that a mutation in a gene (REC104) required for initiation of exchange is completely epistatic to a mutation in RED1. Finally, mutations in either HOP1 or RED1 reduce the number of double-strand breaks observed at the HIS2 meiotic recombination hotspot.     

REC, Drosophila MCM8, drives formation of meiotic crossovers

Crossovers ensure the accurate segregation of homologous chromosomes from one another during meiosis. Here, we describe the identity and function of the Drosophila melanogaster gene recombination defective (rec), which is required for most meiotic crossing over. We show that rec encodes a member of the mini-chromosome maintenance (MCM) protein family. Six MCM proteins (MCM2–7) are essential for DNA replication and are found in all eukaryotes. REC is the Drosophila ortholog of the recently identified seventh member of this family, MCM8. Our phylogenetic analysis reveals the existence of yet another family member, MCM9, and shows that MCM8 and MCM9 arose early in eukaryotic evolution, though one or both have been lost in multiple eukaryotic lineages. Drosophila has lost MCM9 but retained MCM8, represented by REC. We used genetic and molecular methods to study the function of REC in meiotic recombination. Epistasis experiments suggest that REC acts after the Rad51 ortholog SPN-A but before the endonuclease MEI-9. Although crossovers are reduced by 95% in rec mutants, the frequency of noncrossover gene conversion is significantly increased. Interestingly, gene conversion tracts in rec mutants are about half the length of tracts in wild-type flies. To account for these phenotypes, we propose that REC facilitates repair synthesis during meiotic recombination. In the absence of REC, synthesis does not proceed far enough to allow formation of an intermediate that can give rise to crossovers, and recombination proceeds via synthesis-dependent strand annealing to generate only noncrossover products.     

The marine red alga Chondrus crispus has a highly divergent β-tubulin gene with a characteristic 5′ intron: functional and evolutionary implications

We characterized a nuclear gene and its corresponding cDNA encoding β-tubulin (gene TubB1) of the marine red alga Chondrus crispus. The deduced TubB1 protein is the most divergent β-tubulin so far reported with only 64 to 69% amino acid identity relative to other β-tubulins from higher and lower eukaryotes. Our analysis reveals that TubB1 has an accelerated evolutionary rate probably due to a release of functional constraints in connexion with a specialization of microtubular structures in rhodophytes. It further indicates that isoform diversity and functional differentiation of tubulins in eukaryotic cells may be controlled by independent selective constraints. TubB1 has a short spliceosomal intron at its 5′ end which seems to be a characteristic feature of nuclear protein-coding genes from rhodophytes. The splice junctions of the four known rhodophyte introns comply well with the corresponding consensus sequences of higher plants in agreement with previous suggestions from phylogenetic inference that red algae and green plants may be sister groups. The paucity and asymmetrical location of introns in rhodophyte genes can be explained by differential intron loss due to conversion of genes by homologous recombination with cDNAs corresponding to reverse transcribed mRNAs or partially spliced pre-mRNAs, respectively. The identification of an intron containing TubB1 cDNA in C. crispus confirms that pre-mRNAs can escape both splicing and degradation in the nucleus prior to transport into the cytoplasm. Differential Southern hybridizations under non-stringent conditions with homologous and heterologous probes suggest that C. crispus contains a second degenerate β-tubulin gene (or pseudogene?) which, however, is only distantly related to TubB1 as it is to the more conserved homologues of other organisms.     

Molecular and genetic analysis of the yeast early meiotic recombination genes REC102 and REC107/MER2.

By selecting for mutations which could rescue the meiotic lethality of a rad52 spo13 strain, we isolated several new Rec genes required relatively early in the meiotic recombination process. This paper presents data to confirm that two of them, REC102 and REC107, are general, meiosis-specific recombination genes that have no detectable role during mitosis. Sequence analysis and genetic complementation indicate that REC107 is identical to the MER2 gene. No sequences related to REC102 have been found in the GenBank or EMBL collections. REC102 is expressed only in meiosis, prior to the reductional division, at about the time that genetic recombination occurs. Examination of the REC102 sequence indicates the presence of several sequences which may play a role in the regulation of its expression; however, the URS1 sequence commonly found in genes expressed early in meiosis is not present.     

Coordination of the initiation of recombination and the reductional division in meiosis in Saccharomyces cerevisiae

Early exchange (EE) genes are required for the initiation of meiotic recombination in Saccharomyces cerevisiae. Cells with mutations in several EE genes undergo an earlier reductional division (MI), which suggests that the initiation of meiotic recombination is involved in determining proper timing of the division. The different effects of null mutations on the timing of reductional division allow EE genes to be assorted into three classes: mutations in RAD50 or REC102 that confer a very early reductional division; mutations in REC104 or REC114 that confer a division earlier than that of wild-type (WT) cells, but later than that of mutants of the first class; and mutations in MEI4 that do not significantly alter the timing of MI. The very early mutations are epistatic to mutations in the other two classes. We propose a model that accounts for the epistatic relationships and the communication between recombination initiation and the first division. Data in this article indicate that double-strand breaks (DSBs) are not the signal for the normal delay of reductional division; these experiments also confirm that MEI4 is required for the formation of meiotic DSBs. Finally, if a DSB is provided by the HO endonuclease, recombination can occur in the absence of MEI4 and REC104.     

Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4

The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of thetd gene by recombinational marker rescue using plasmid or M13 subclones of thetd gene. Phage crosses using deletion mutants with known breakpoints in the 3′ end of thetd intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5′ and 3′ ends of the intron sequence, in accordance with the hypothesis that the 5′ and 3′ domains of the T4td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mappedtd mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within thetd gene.     

Evolutionary and Functional Analysis of the Invariant SWIM Domain in the Conserved Shu2/SWS1 Protein Family from Saccharomyces cerevisiae to Homo sapiens

The Saccharomyces cerevisiae Shu2 protein is an important regulator of Rad51, which promotes homologous recombination (HR). Shu2 functions in the Shu complex with Shu1 and the Rad51 paralogs Csm2 and Psy3. Shu2 belongs to the SWS1 protein family, which is characterized by its SWIM domain (CXC...X_n...CXH), a zinc-binding motif. In humans, SWS1 interacts with the Rad51 paralog SWSAP1. Using genetic and evolutionary analyses, we examined the role of the Shu complex in mitotic and meiotic processes across eukaryotic lineages. We provide evidence that the SWS1 protein family contains orthologous genes in early-branching eukaryote lineages (e.g., Giardia lamblia), as well as in multicellular eukaryotes including Caenorhabditis elegans and Drosophila melanogaster. Using sequence analysis, we expanded the SWIM domain to include an invariant alanine three residues after the terminal CXH motif (CXC…X_n…CXHXXA). We found that the SWIM domain is conserved in all eukaryotic orthologs, and accordingly, in vivo disruption of the invariant residues within the canonical SWIM domain inhibits DNA damage tolerance in yeast and protein-protein interactions in yeast and humans. Furthermore, using evolutionary analyses, we found that yeast and Drosophila Shu2 exhibit strong coevolutionary signatures with meiotic proteins, and in yeast, its disruption leads to decreased meiotic progeny. Together our data indicate that the SWS1 family is an ancient and highly conserved eukaryotic regulator of meiotic and mitotic HR.