Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments

Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.     

The mouse Spo11 gene is required for meiotic chromosome synapsis

The Spo11 protein initiates meiotic recombination by generating DNA double-strand breaks (DSBs) and is required for meiotic synapsis in S. cerevisiae. Surprisingly, Spo11 homologs are dispensable for synapsis in C. elegans and Drosophila yet required for meiotic recombination. Disruption of mouse Spo11 results in infertility. Spermatocytes arrest prior to pachytene with little or no synapsis and undergo apoptosis. We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis. Spo11 localizes to discrete foci during leptotene and to homologously synapsed chromosomes. Other mouse mutants that arrest during meiotic prophase (Atm -/-, Dmc1 -/-, mei1, and Morc(-/-)) showed altered Spo11 protein localization and expression. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis.     

The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion.     

Meiotic Crossover Control by Concerted Action of Rad51-Dmc1 in Homolog Template Bias and Robust Homeostatic Regulation

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1''s strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51''s strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1''s chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1''s dominance in promoting strand exchange between homologs involves repression of Rad51''s strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination.     

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.     

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.     

Roles of RecA homologues Rad51 and Dmc1 during meiotic recombination

RecA protein is involved in homology search and strand exchange processes during recombination. Mitotic cells in eukaryotes express one RecA, Rad51, which is essential for the repair of double-strand breaks (DSBs). Additionally, meiotic cells induce the second RecA, Dmc1. Both Rad51 and Dmc1 are necessary to generate a crossover between homologous chromosomes, which ensures the segregation of the chromosomes at meiotic division I. It is largely unknown how the two RecAs cooperate during meiotic recombination. In this review, recent advances on our knowledge about the roles of Rad51 and Dmc1 during meiosis are summarized and discussed.     

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.     

Recombinational repair in Schizosaccharomyces pombe: role in maintaining genomic integrity

Recombinational repair was first detected in budding yeast Saccharomyces cerevisiae and was also studied in fission yeast Schizosaccharomyces pombe over the recent decade. The discovery of Sch. pombe homologs of the S. cerevisiae RAD52 genes made it possible not only to identify and to clone their vertebrate counterparts, but also to study in detail the role of DNA recombination in certain cell processes. For instance, recombinational repair was shown to play a greater role in maintaining genome integrity in fission yeast and in vertebrates compared with S. cerevisiae. The present state of the problem of recombinational double-strand break repair in fission yeast is considered with a focus on comparisons between Sch. pombe and higher eukaryotes. The role of double-strand break repair in maintaining genome stability is discussed.     

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.     

Dmc1 Functions in a Saccharomyces Cerevisiae Meiotic Pathway That Is Largely Independent of the Rad51 Pathway

Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p. A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype. A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D. Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p. Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p. However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes. Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination. Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin. Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains.     

The recombinases Rad51 and Dmc1 play distinct roles in DNA break repair and recombination partner choice in the meiosis of Tetrahymena

Repair of programmed DNA double-strand breaks (DSBs) by meiotic recombination relies on the generation of flanking 3' single-stranded DNA overhangs and their interaction with a homologous double-stranded DNA template. In various common model organisms, the ubiquitous strand exchange protein Rad51 and its meiosis-specific homologue Dmc1 have been implicated in the joint promotion of DNA-strand exchange at meiotic recombination sites. However, the division of labor between these two recombinases is still a puzzle. Using RNAi and gene-disruption experiments, we have studied their roles in meiotic recombination and chromosome pairing in the ciliated protist Tetrahymena as an evolutionarily distant meiotic model. Cytological and electrophoresis-based assays for DSBs revealed that, without Rad51p, DSBs were not repaired. However, in the absence of Dmc1p, efficient Rad51p-dependent repair took place, but crossing over was suppressed. Immunostaining and protein tagging demonstrated that only Dmc1p formed strong DSB-dependent foci on meiotic chromatin, whereas the distribution of Rad51p was diffuse within nuclei. This suggests that meiotic nucleoprotein filaments consist primarily of Dmc1p. Moreover, a proximity ligation assay confirmed that little if any Rad51p forms mixed nucleoprotein filaments with Dmc1p. Dmc1p focus formation was independent of the presence of Rad51p. The absence of Dmc1p did not result in compensatory assembly of Rad51p repair foci, and even artificial DNA damage by UV failed to induce Rad51p foci in meiotic nuclei, while it did so in somatic nuclei within one and the same cell. The observed interhomologue repair deficit in dmc1Δ meiosis is consistent with a requirement for Dmc1p in promoting the homologue as the preferred recombination partner. We propose that relatively short and/or transient Rad51p nucleoprotein filaments are sufficient for intrachromosomal recombination, whereas long nucleoprotein filaments consisting primarily of Dmc1p are required for interhomolog recombination.     

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.     

Mnd1/Hop2 facilitates Dmc1-dependent interhomolog crossover formation in meiosis of budding yeast

During meiosis, each chromosome must pair with its homolog and undergo meiotic crossover recombination in order to segregate properly at the first meiotic division. Recombination in meiosis in Saccharomyces cerevisiae relies on two Escherichia coli recA homologs, Rad51 and Dmc1, as well as the more recently discovered heterodimer Mnd1/Hop2. Meiotic recombination in S. cerevisiae mnd1 and hop2 single mutants is initiated via double-strand breaks (DSBs) but does not progress beyond this stage; heteroduplex DNA, joint molecules, and crossovers are not detected. Whereas hop2 and mnd1 single mutants are profoundly recombination defective, we show that mnd1 rad51, hop2 rad51, and mnd1 rad17 double mutants are able to carry out crossover recombination. Interestingly, noncrossover recombination is absent, indicating a role for Mnd1/Hop2 in the designation of DSBs for noncrossover recombination. We demonstrate that in the rad51 mnd1 double mutant, recombination is more likely to occur between repetitive sequences on nonhomologous chromosomes. Our results support a model in which Mnd1/Hop2 is required for DNA-DNA interactions that help ensure Dmc1-mediated stable strand invasion between homologous chromosomes, thereby preserving genomic integrity.     

Saccharomyces cerevisiae Dmc1 protein promotes renaturation of single-strand DNA (ssDNA) and assimilation of ssDNA into homologous super-coiled duplex DNA

Dmc1 and Rad51 are eukaryotic RecA homologues that are involved in meiotic recombination. The expression of Dmc1 is limited to meiosis, whereas Rad51 is expressed in mitosis and meiosis. Dmc1 and Rad51 have unique and overlapping functions during meiotic recombination. Here we report the purification of the Dmc1 protein from the budding yeast Saccharomyces cerevisiae and present basic characterization of its biochemical activity. The protein has a weak DNA-dependent ATPase activity and binds both single-strand DNA (ssDNA) and double-strand DNA. Electrophoretic mobility shift assays suggest that DNA binding by Dmc1 is cooperative. Dmc1 renatures linearized plasmid DNA with first order reaction kinetics and without requiring added nucleotide cofactor. In addition, Dmc1 catalyzes strand assimilation of ssDNA oligonucleotides into homologous supercoiled duplex DNA in a reaction promoted by ATP or the non-hydrolyzable ATP analogue AMP-PNP.     

Cell cycle-regulated centers of DNA double-strand break repair

In eukaryotes, homologous recombination is an important pathway for the repair of DNA double-strand breaks. We have studied this process in living cells in the yeast Saccharomyces cerevisiae using Rad52 as a cell biological marker. In response to DNA damage, Rad52 redistributes itself and forms foci specifically during S phase. We have shown previously that Rad52 foci are centers of DNA repair where multiple DNA double-strand breaks colocalize. Here we report a correlation between the timing of Rad52 focus formation and modification of the Rad52 protein. In addition, we show that the two ends of a double-strand break are held tightly together in the majority of cells. Interestingly, in a small but significant fraction of the S phase cells, the two ends of a break separate suggesting that mechanisms exist to reassociate and align these ends for proper DNA repair.     

Saccharomyces cerevisiae Dmc1 and Rad51 Proteins Preferentially Function with Tid1 and Rad54 Proteins, Respectively, to Promote DNA Strand Invasion during Genetic Recombination

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.     

Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.

The RAD51 gene of S. cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis. We show that the rad51 null mutant accumulates meiosis-specific double-strand breaks (DSBs) at a recombination hotspot and reduces the formation of physical recombinants. Rad51 protein shows structural similarity to RecA protein, the bacterial strand exchange protein. Furthermore, we have found that Rad51 protein is similar to RecA in its DNA binding properties and binds directly to Rad52 protein, which also plays a crucial role in recombination. These results suggest that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination. Rad51 protein is also homologous to a meiosis-specific Dmc1 protein of S. cerevisiae.     

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.