Initiation of meiotic recombination in the yeast

The Saccharomyces cerevisiae DBR1 gene encodes a 2-5 phosphodiesterase that debranches intron RNA lariats following splicing. Yeast dbr1 mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3. We used a mutagenic PCR method to generate a collection of dbr1 mutant alleles to explore the relationship between the roles of DBR1 in transposition and debranching. Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p. Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition. Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine. We have developed an RNase protection assay to quantitate intron RNA accumulation in cells. The assay uses RNA probes that hybridize to ACT1 intron RNA. Protection patterns confirm that sequences from the 5 end of the intron to the lariat branch point accumulate in dbr1 mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generated dbr1 mutant alleles are also deficient for debranching, further supporting a role for 2-5 phosphodiesterase activity in Ty1 transposition. A Ty1 element lacking most of its internal sequences transposes independently of DBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.Saccharomyces cerevisiae Ty1 is a long terminal repeat (LTR) retroelement with a life cycle similar to that of retroviruses (2). Like retroviruses, Ty1 replicates via reverse transcription of an RNA intermediate, subsequently integrating element cDNA into the host genome. Retroviruses and LTR retrotransposons rely on their hosts to provide most of the factors necessary for propagation. We identified dbr1 mutants in a screen to identify host cell genes required for Ty1 transposition (15). The dbr1 mutant had been identified previously in a similar screen by others (6). Although dbr1 cells produce wild-type levels of Ty1 proteins, they accumulate Ty1 cDNA at a slower rate than do wild-type cells, suggesting that Dbr1p plays a role in Ty1 reverse transcription or cDNA stability (15).In its cellular role, Dbr1p acts at the end of the mRNA splicing process, cleaving the 2Ј-5Ј linkage at the branch point of intron RNA lariats released after exon ligation, converting them to linear RNAs that are rapidly degraded (6, 23). In a dbr1 mutant, intron RNAs accumulate as lariats (6). The uncleaved lariat branch point blocks the progression of 3Ј exonucleases, and there is no 5Ј end available on which a 5Ј exonuclease can act. DBR1 is highly conserved, and homologues have been cloned from Schizosaccharomyces pombe, S. cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens (6,17,18,24). Deletion of DBR1 has no significant effect on the growth rate of S. cerevisiae, but...

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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.

REC104 is a gene required for the initiation of meiotic recombination in Saccharomyces cerevisiae. To better understand the role of REC104 in meiosis, we used an in vitro mutagenesis technique to create a set of temperature-conditional mutations in REC104 and used one ts allele (rec104-8) in a screen for highcopy suppressors. An increased dosage of the early exchange gene REC102 was found to suppress the conditional recombinational reduction in rec104-8 as well as in several other conditional rec104 alleles. However, no suppression was observed for a null allele of REC104, indicating that the suppression by REC102 is not “bypass” suppression. Overexpression of the early meiotic genes REC114, RAD50, HOP1, and RED1 fails to suppress any of the rec104 conditional alleles, indicating that the suppression might be specific to REC102.