Robert E. Malone

The RAD50 gene, a member of the double strand break repair epistasis group, is not required for spontaneous mitotic recombination in yeast

SummaryMutations in the RAD50 gene of Saccharomyces cerevisiae have been shown to reduce double strand break repair, meiotic recombination, and radiation-inducible mitotic recombination. Several different point mutations (including ochre and amber alleles) have been previously examined for effects on spontaneous mitotic recombination and did not reduce the frequency of recombination. Instead, the rad50 mutations conferred a moderate hyper-rec phenotype. This paper examines a deletion/interruption allele of RAD50 that removes 998 of 1312 amino acids and adds 1.1 kb of foreign DNA. The results clearly indicate that spontaneous mitotic recombination can occur in the absence of RAD50; in fact, the frequency of recombination is elevated over the wild-type cell. One possible interpretation of these observations is that the initiating lesion in spontaneous recombination events in mitosis might not be a double strand break.

Dual regulation of meiosis in yeast.

The two regulatory pathways appear to come together at the IME1 gene. It is clearly regulated by mating type and induced by starvation as well. Overexpression of IME1 completely overcomes MAT defects but may not circumvent all nutritional control. Kassir et al. (1988) found that overexpression of IME1 allowed sporulation in the presence of glucose and nitrogen. They also have found a meiotic level of message in temperature-sensitive cdc25 diploids shifted to high temperature in rich medium (Simchen and Kassir, 1989). Smith and Mitchell (1989) found that overexpression of IME1 induced an early meiotic event (recombination) in rich medium, but later meiotic events did not occur (i.e., they detected no spore formation). Mitchell (personal communication) has suggested that the difference may be due to differences in the amount of nitrogen present in the two experiments. Thus, while it is clear that IME1 is a necessary positive regulator of meiosis, responding both to mating type and nutritional conditions, it is not clear if it is sufficient. It is possible that other genes are involved in the response to starvation. One interpretation is that a separate nutritional control is exerted for events starting with meiosis I. Much of the regulatory pathway that allows yeast cells to enter meiosis has been determined. As in the case in many sensory transduction pathways, the initial signal for starvation is not yet known, nor is the nature of the proposed downstream phosphorylated effector. Given the power of yeast molecular genetics, answers to both these questions seem attainable. Another area that remains unclear is the difference between responses to nitrogen starvation versus carbon source. Many of the experiments discussed above do not address this question. The strategies used by yeast may be utilized in the developmental decisions used by other, more complex eukaryotes. Certainly several of the gene products involved in nutritional control in yeast have homologies in mammalian systems. For example, the human H-ras gene can substitute for yeast RAS; the relationship is sufficiently close that dominant Ha-ras mutations that inhibit CDC25 have been found (Powers et al., 1989). Furthermore, these dominant Ha-ras mutations have the appropriate phenotype in mammalian cells, suggesting the presence of a CDC25-like protein. Although the major components of mating type control appear to have been defined, the mechanism of the RME1-IME transcriptional control remains to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)

A reexamination of the role of the RAD52 gene in spontaneous mitotic recombination

SummaryThe RAD52 gene is required for much of the recombination that occurs in Saccharomyces cerevisiae. One of the two commonly utilized mutant alleles, rad52-2, increases rather than reduces mitotic recombination, yet in other respects appears to be a typical rad52 mutant allele. This raises the question as to whether RAD52 is really necessary for mitotic recombination. Analysis of a deletion/insertion allele created in vitro indicates that the null mutant phenotype is indeed a deficiency in mitotic recombination, especially in gene conversion. The data also indicate that RAD52 is required for crossing-over between at least some chromosomes. Finally, examination of the behavior of a replicating plasmid in rad52-1 strains indicates that the frequency of plasmid integration is substantially reduced from that in wild type, a conclusion consistent with a role for RAD52 in reciprocal crossing-over. Analysis of recombinants arising in rad52-2 strains suggests that this allele may result in the increased activity of a RAD52-independent recombinational pathway.

Support for a Meiotic Recombination Initiation Complex: Interactions among Rec102p, Rec104p, and Spo11p

ABSTRACT Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae requires at least 10 gene products. The initiation event creates double-strand breaks, which are then processed by other recombination enzymes. A variety of classical observations, such as the existence of recombination nodules, have suggested that the proteins catalyzing recombination form a complex. A variety of lines of evidence indicate that Rad50p, Mre11p, and Xrs2p interact, and genetic data suggesting interactions between Rec102p and Rec104p have been reported. It has recently been shown that Spo11p coimmunoprecipitates with Rec102p in meiosis as well. In this paper, we provide genetic and biochemical evidence that the meiosis-specific proteins Rec102p, Rec104p, and Spo11p all interact with each other in meiosis. Furthermore, we demonstrate that the interaction between Rec102p and Spo11p does not require Rec104p. Likewise, the interaction between Rec104p and Rec102p does not require Spo11p, although Spo11p may stabilize that association. The interactions suggest that Spo11p, Rec102p, and Rec104p may form a trimeric complex during the initiation of recombination.

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.

The Signal from the Initiation of Meiotic Recombination to the First Division of Meiosis

ABSTRACT Two of the unique events that occur in meiosis are high levels of genetic recombination and the reductional division. Our previous work demonstrated that the REC102, REC104, REC114, and RAD50 genes, required to initiate meiotic recombination in Saccharomyces cerevisiae, are needed for the proper timing of the first meiotic (MI) division. If these genes are absent, the MI division actually begins at an earlier time. This paper demonstrates that the meiotic recombination genes MER2/REC107, SPO11, and MRE2 and the synaptonemal complex genes HOP1 and RED1 are also required for the normal delay of the MI division. A rec103/ski8 mutant starts the MI division at the same time as in wild-type cells. Our data indicate no obvious correlation between the timing of premeiotic S phase and the timing of the first division in Rec− mutants. Cells with rec102 or rec104 mutations form MI spindles before wild-type cells, suggesting that the initiation signal acts prior to spindle formation. Neither RAD9 nor RAD24 is needed to transduce the signal, which delays the first division. The timing of the MI division in RAD24 mutants indicates that the pachytene checkpoint is not active in Rec+ cells and suggests that the coordination between recombination and the MI division in wild-type cells may occur primarily due to the initiation signal. Finally, at least one of the targets of the recombination initiation signal is the NDT80 gene, a transcriptional regulator of middle meiotic gene expression required for the first division.

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.

Genetic and molecular analysis of REC114, an early meiotic recombination gene in yeast

Four new meiotic recombination genes were previously isolated by selecting for mutations that rescue the meiotic lethality of rad52 spo13 strains. One of these genes, REC114, is described here, and the data confirm that REC114 is a meiosis-specific recombination gene with no detectable function in mitosis. REC114 is located on chromosome XIII approximately 4,9 cM from CIN4. The nucleotide sequence reveals an open reading frame of 1262 bp, consensus intron splice sites close to the 3′ end, and indicates that the second exon codes for only seven amino acids. In the promoter region, a URS1 consensus sequence (TGGGCGGCTA), identical to the URS1 found in the promoter of SPO16, is present 93 bp upstream of the translation start site. Northern-blot hybridization demonstrates that REC114 is transcribed only during meiosis and that it is not expressed in the absence of the IME1 gene product, even when IME2 is constitutively expressed.

A test of the CoHR motif associated with meiotic double-strand breaks in Saccharomyces cerevisiae

Meiotic recombination is not random along chromosomes; rather, there are preferred regions for initiation called hotspots. Although the general properties of meiotic hotspots are known, the requirements at the DNA sequence level for the determination of hotspot activity are still unclear. The sequence of six known hotspots in Saccharomyces cerevisiae was compared to identify a common homology region (CoHR). They reported that the locations of CoHR sequences correspond to mapped double‐strand break (DSB) sites along three chromosomes (I, III, VI). We report here that a deletion of CoHR at HIS2, a hotspot used to identify the motif, has no significant effect on recombination. In the absence of CoHR, DSB formation occurs at a high frequency and at the same sequences as in wild‐type strains. In cases where the deletion of sequences containing the CoHR motif has been shown to reduce recombination, we propose that it may be a reflection of the location of the deletion, rather than the loss of CoHR, per se.

Phylogenetic footprinting reveals multiple regulatory elements involved in control of the meiotic recombination gene, REC102

REC102 is a meiosis‐specific early exchange gene absolutely required for meiotic recombination in Saccharomyces cerevisiae. Sequence analysis of REC102 indicates that there are multiple potential regulatory elements in its promoter region, and a possible regulatory element in the coding region. This suggests that the regulation of REC102 may be complex and may include elements not yet reported in other meiotic genes. To identify potential cis‐regulatory elements, phylogenetic footprinting analysis was used. REC102 homologues were cloned from other two Saccharomyces spp. and sequence comparison among the three species defined evolutionarily conserved elements. Deletion analysis demonstrated that the early meiotic gene regulatory element URS1 was necessary but not sufficient for proper regulation of REC102. Upstream elements, including the binding sites for Gcr1p, Yap1p, Rap1p and several novel conserved sequences, are also required for the normal regulation of REC102 as well as a Rap1p binding site located in the coding region. The data in this paper support the use of phylogenetic comparisions as a method for determining important sequences in complex promoters. Copyright