Down-Regulation of Rad51 Activity during Meiosis in Yeast Prevents Competition with Dmc1 for Repair of Double-Strand Breaks

Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs.     

The Rad52 homologs Rad22 and Rti1 of Schizosaccharomyces pombe are not essential for meiotic interhomolog recombination, but are required for meiotic intrachromosomal recombination and mating-type-related DNA repair

Proteins of the RAD52 epistasis group play an essential role in repair of some types of DNA damage and genetic recombination. In Schizosaccharomyces pombe, Rad22 (a Rad52 ortholog) has been shown to be as necessary for repair and recombination events during vegetative growth as its Saccharomyces cerevisiae counterpart. This finding contrasts with previous reports where, due to suppressor mutations in the fbh1 gene, rad22 mutants did not display a severe defect. We have analyzed the roles of Rad22 and Rti1, another Rad52 homolog, during meiotic recombination and meiosis in general. Both proteins play an important role in spore viability. During meiotic prophase I, they partially colocalize and partially localize to Rad51 foci and linear elements. Genetic analysis showed that meiotic interchromosomal crossover and conversion events were unexpectedly not much affected by deletion of either or both genes. A strong decrease of intrachromosomal recombination assayed by a gene duplication construct was observed. Therefore, we propose that the most important function of Rad22 and Rti1 in S. pombe meiosis is repair of double-strand breaks with involvement of the sister chromatids. In addition, a novel mating-type-related repair function of Rad22 specific to meiosis and spore germination is described.     

Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis.     

Phosphorylation of the axial element protein Hop1 by Mec1/Tel1 ensures meiotic interhomolog recombination

An essential feature of meiosis is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks (DSBs) is repaired using an intact homologous non-sister chromatid rather than a sister. Involvement of Mec1 and Tel1, the budding yeast homologs of the mammalian ATR and ATM kinases, in meiotic interhomlog bias has been implicated, but the mechanism remains elusive. Here, we demonstrate that Mec1 and Tel1 promote meiotic interhomolog recombination by targeting the axial element protein Hop1. Without Mec1/Tel1 phosphorylation of Hop1, meiotic DSBs are rapidly repaired via a Dmc1-independent intersister repair pathway, resulting in diminished interhomolog crossing-over leading to spore lethality. We find that Mec1/Tel1-mediated phosphorylation of Hop1 is required for activation of Mek1, a meiotic paralogue of the DNA-damage effector kinase, Rad53p/CHK2. Thus, Hop1 is a meiosis-specific adaptor protein of the Mec1/Tel1 signaling pathway that ensures interhomolog recombination by preventing Dmc1-independent repair of meiotic DSBs.     

The<Emphasis Type="Italic">atspo11-1</Emphasis> mutation rescues <Emphasis Type="Italic">atxrcc3</Emphasis> meiotic chromosome fragmentation

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division. These events are initiated by programmed double-strand breaks produced by the Spo11 protein and repair of such breaks by homologous recombination requires a strand exchange activity provided by the Rad51 protein. We have recently reported that the absence of AtXrcc3, an ArabidopsisRad51 paralogue, leads to extensive chromosome fragmentation during meiosis, first visible in diplotene of meiotic prophase I. The present study clearly shows that this fragmentation results from un- or mis-repaired AtSpo11-1 induced double-strand breaks and is thus due to a specific defect in the meiotic recombination process.     

Elevated Rad53 kinase activity influences formation and interhomolog repair of meiotic DNA double-strand breaks in budding yeast

Meiotic cells generate physiological programmed DNA double-strand breaks (DSBs) to initiate meiotic recombination. Interhomolog repair of the programmed DSBs by meiotic recombination is vital to ensure accurate chromosome segregation at meiosis I to produce normal gametes. In budding yeast, the DNA damage checkpoint kinase Rad53 is activated by DSBs which accidentally occur as DNA lesions in mitosis and meiosis; however, meiotic programmed DSBs which occur at ∼160 loci per genome fail to activate the kinase. Thus, Rad53 activation appears to be silenced in response to meiotic programmed DSBs. In this study, to address the biological significance of Rad53’s insensitivity to meiotic DSBs, we examined the effects of Rad53 overexpression on meiotic processes. The overexpression led to partial activation of Rad53, uncovering that the negative impacts of Rad53 kinase activation on meiotic progression, and formation and interhomolog repair of meiotic programmed DSBs.     

The mitotic DNA damage checkpoint proteins Rad17 and Rad24 are required for repair of double-strand breaks during meiosis in yeast

We show here that deletion of the DNA damage checkpoint genes RAD17 and RAD24 in Saccharomyces cerevisiae delays repair of meiotic double-strand breaks (DSBs) and results in an altered ratio of crossover-to-noncrossover products. These mutations also decrease the colocalization of immunostaining foci of the RecA homologs Rad51 and Dmc1 and cause a delay in the disappearance of Rad51 foci, but not of Dmc1. These observations imply that RAD17 and RAD24 promote efficient repair of meiotic DSBs by facilitating proper assembly of the meiotic recombination complex containing Rad51. Consistent with this proposal, extra copies of RAD51 and RAD54 substantially suppress not only the spore inviability of the rad24 mutant, but also the gamma-ray sensitivity of the mutant. Unexpectedly, the entry into meiosis I (metaphase I) is delayed in the checkpoint single mutants compared to wild type. The control of the cell cycle in response to meiotic DSBs is also discussed.     

Schizosaccharomyces pombe rad32 protein: a phosphoprotein with an essential phosphoesterase motif required for repair of DNA double strand breaks.

The Schizosaccharomyces pombe Rad32 protein is required for repair of DNA double strand breaks, minichromosome stability and meiotic recombination. We show here that the Rad32 protein is phosphorylated in a cell cycle-dependent manner and during meiosis. The phosphorylation is not dependent on the checkpoint protein Rad3. Analysis of a partially purified protein preparation indicates that Rad32 is likely to act in a complex. Characterisation of the rad32-1 mutation and site-directed mutagenesis indicate that three aspartate residues in the conserved phosphoesterase motifs are important for both mitotic and meiotic functions, namely response to UV and ionising radiation and spore viability.     

Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1

During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.     

EXO1 and MSH4 differentially affect crossing-over and segregation

The 5′-3′ exonuclease Exo1p from Saccharomyces cerevisiae is required for wild-type levels of meiotic crossing-over and normal meiotic chromosome segregation as is the meiosis-specific MutS homologue, Msh4p. Mutations in both genes reduce crossing-over by approximately two-fold, but Δmsh4 strains have significantly lower viability and a higher frequency of meiosis I non-disjunction. Epistasis analysis indicates a complex interaction between the two genes. Although crossing-over was not detectably lower in the double mutant, viability was significantly worse than either single mutant. Such a result suggests that the two genes are affecting meiotic viability by distinct mechanisms. We propose that Δexo1 affects chromosome segregation by reducing crossing-over, while Δmsh4 affects both the frequency and distribution of crossovers. Mutation in EXO1 reduces gene conversion frequencies significantly at some but not all loci, suggesting that other enzymes are also involved in DNA resection. We propose that Exo1p plays an early role in establishing some recombination intermediates by generating single-stranded tails. The role of Msh4p is suggested to be in determining whether some recombination intermediates are resolved as crossover events and in generating crossover interference. The synergistic effect of Δexo1Δmsh4 on spore viability suggests that the two genes have partially compensatory roles in a process affecting meiotic success. Received: 10 November 1999; in revised form: 14 January 2000 / Accepted: 14 January 2000     

Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI)

During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.     

Sustained and rapid chromosome movements are critical for chromosome pairing and meiotic progression in budding yeast

Telomere-led chromosome movements are a conserved feature of meiosis I (MI) prophase. Several roles have been proposed for such chromosome motion, including promoting homolog pairing and removing inappropriate chromosomal interactions. Here, we provide evidence in budding yeast that rapid chromosome movements affect homolog pairing and recombination. We found that csm4Δ strains, which are defective for telomere-led chromosome movements, show defects in homolog pairing as measured in a "one-dot/two-dot tetR-GFP" assay; however, pairing in csm4Δ eventually reaches near wild-type (WT) levels. Charged-to-alanine scanning mutagenesis of CSM4 yielded one allele, csm4-3, that confers a csm4Δ-like delay in meiotic prophase but promotes high spore viability. The meiotic delay in csm4-3 strains is essential for spore viability because a null mutation (rad17Δ) in the Rad17 checkpoint protein suppresses the delay but confers a severe spore viability defect. csm4-3 mutants show a general defect in chromosome motion but an intermediate defect in chromosome pairing. Chromosome velocity analysis in live cells showed that while average chromosome velocity was strongly reduced in csm4-3, chromosomes in this mutant displayed occasional rapid movements. Lastly, we observed that spo11 mutants displaying lower levels of meiosis-induced double-strand breaks showed higher spore viability in the presence of the csm4-3 mutation compared to csm4Δ. On the basis of these observations, we propose that during meiotic prophase the presence of occasional fast moving chromosomes over an extended period of time is sufficient to promote WT levels of recombination and high spore viability; however, sustained and rapid chromosome movements are required to prevent a checkpoint response and promote efficient meiotic progression.     

Increased Meiotic Crossovers and Reduced Genome Stability in Absence of Schizosaccharomyces pombe Rad16 (XPF)

Schizosaccharomyces pombe Rad16 is the ortholog of the XPF structure-specific endonuclease, which is required for nucleotide excision repair and implicated in the single strand annealing mechanism of recombination. We show that Rad16 is important for proper completion of meiosis. In its absence, cells suffer reduced spore viability and abnormal chromosome segregation with evidence for fragmentation. Recombination between homologous chromosomes is increased, while recombination within sister chromatids is reduced, suggesting that Rad16 is not required for typical homolog crossovers but influences the balance of recombination between the homolog and the sister. In vegetative cells, rad16 mutants show evidence for genome instability. Similar phenotypes are associated with mutants affecting Rhp14^XPA but are independent of other nucleotide excision repair proteins such as Rad13^XPG. Thus, the XPF/XPA module of the nucleotide excision repair pathway is incorporated into multiple aspects of genome maintenance even in the absence of external DNA damage.     

S. cerevisiae Srs2 helicase ensures normal recombination intermediate metabolism during meiosis and prevents accumulation of Rad51 aggregates

We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalised with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards interhomologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division.

     

Analysis of close stable homolog juxtaposition during meiosis in mutants of Saccharomyces cerevisiae

A unique aspect of meiosis is the segregation of homologous chromosomes at the meiosis I division. The pairing of homologous chromosomes is a critical aspect of meiotic prophase I that aids proper disjunction at anaphase I. We have used a site-specific recombination assay in Saccharomyces cerevisiae to examine allelic interaction levels during meiosis in a series of mutants defective in recombination, chromatin structure, or intracellular movement. Red1, a component of the chromosome axis, and Mnd1, a chromosome-binding protein that facilitates interhomolog interaction, are critical for achieving high levels of allelic interaction. Homologous recombination factors (Sae2, Rdh54, Rad54, Rad55, Rad51, Sgs1) aid in varying degrees in promoting allelic interactions, while the Srs2 helicase appears to play no appreciable role. Ris1 (a SWI2/SNF2 related protein) and Dot1 (a histone methyltransferase) appear to play minor roles. Surprisingly, factors involved in microtubule-mediated intracellular movement (Tub3, Dhc1, and Mlp2) appear to play no appreciable role in homolog juxtaposition, unlike their counterparts in fission yeast. Taken together, these results support the notion that meiotic recombination plays a major role in the high levels of homolog interaction observed during budding yeast meiosis.     

Meiotic Recombination Initiated by a Double-Strand Break in Rad50Δ Yeast Cells Otherwise Unable to Initiate Meiotic Recombination

Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast's 16 chromosomes. In Rad(+) strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad(+) and in rad50Δ cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50Δ cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad50Δ diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination.     

The requirement for ATP hydrolysis by Saccharomyces cerevisiae Rad51 is bypassed by mating-type heterozygosity or RAD54 in high copy

Rad51 can promote extensive strand exchange in vitro in the absence of ATP hydrolysis, and the Rad51-K191R mutant protein, which can bind but poorly hydrolyze ATP, also promotes strand exchange. A haploid strain expressing the rad51-K191R allele showed an equivalent sensitivity at low doses of ionizing radiation to rad51-K191A or rad51 null mutants and was defective in spontaneous and double-strand break-induced mitotic recombination. However, the rad51-K191R/rad51-K191R diploid sporulated and the haploid spores showed high viability, indicating no apparent defect in meiotic recombination. The DNA repair defect caused by the rad51-K191R allele was suppressed in diploids and by mating-type heterozygosity in haploids. RAD54 expressed from a high-copy-number plasmid also suppressed the gamma-ray sensitivity of rad51-K191R haploids. The suppression by mating-type heterozygosity of the DNA repair defect conferred by the rad51-K191R allele could occur by elevated expression of factors that act to stabilize, or promote catalysis, by the partially functional Rad51-K191R protein.     

Schizosaccharomyces pombe Rdh54 (TID1) acts with Rhp54 (RAD54) to repair meiotic double-strand breaks

We report the characterization of rdh54+, the second fission yeast Schizosaccharomyces pombe Rad54 homolog. rdh54+ shares sequence and functional homology to budding yeast RDH54/TID1. Rdh54p is present during meiosis with appropriate timing for a meiotic recombination factor. It interacts with Rhp51 and the meiotic Rhp51 homolog Dmc1 in yeast two-hybrid assays. Deletion of rdh54+ has no effect on DNA damage repair during the haploid vegetative cell cycle. In meiosis, however, rdh54Delta shows decreased spore viability and homologous recombination with a concomitant increase in sister chromatid exchange. The rdh54Delta single mutant repairs meiotic breaks with similar timing to wild type, suggesting redundancy of meiotic recombination factors. Consistent with this, the rdh54Delta rhp54Delta double mutant fails to repair meiotic double strand breaks. Live cell analysis shows that rdh54Delta rhp54Delta asci do not arrest, but undergo both meiotic divisions with near normal timing, suggesting that failure to repair double strand breaks in S. pombe meiosis does not result in checkpoint arrest.     

Crossover interference in Saccharomyces cerevisiae requires a TID1/RDH54- and DMC1-dependent pathway

Two RecA-like recombinases, Rad51 and Dmc1, function together during double-strand break (DSB)-mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae. Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors. Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type. Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis. Previous work showed that dmc1Delta mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54. Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Delta cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective. These results support the proposal that crossover interference acts at the strand invasion stage of recombination.     

Novel attributes of Hed1 affect dynamics and activity of the Rad51 presynaptic filament during meiotic recombination

During meiosis, recombination events that occur between homologous chromosomes help prepare the chromosome pairs for proper disjunction in meiosis I. The concurrent action of the Rad51 and Dmc1 recombinases is necessary for an interhomolog bias. Notably, the activity of Rad51 is tightly controlled, so as to minimize the use of the sister chromatid as recombination partner. We demonstrated recently that Hed1, a meiosis-specific protein in Saccharomyces cerevisiae, restricts the access of the recombinase accessory factor Rad54 to presynaptic filaments of Rad51. We now show that Hed1 undergoes self-association in a Rad51-dependent manner and binds ssDNA. We also find a strong stabilizing effect of Hed1 on the Rad51 presynaptic filament. Biochemical and genetic analyses of mutants indicate that these Hed1 attributes are germane for its recombination regulatory and Rad51 presynaptic filament stabilization functions. Our results shed light on the mechanism of action of Hed1 in meiotic recombination control.