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.     

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.     

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.     

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.     

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.     

Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1

Two BRCA2-like sequences are present in the Arabidopsis genome. Both genes are expressed in flower buds and encode nearly identical proteins, which contain four BRC motifs. In a yeast two-hybrid assay, the Arabidopsis Brca2 proteins interact with Rad51 and Dmc1. RNAi constructs aimed at silencing the BRCA2 genes at meiosis triggered a reproducible sterility phenotype, which was associated with dramatic meiosis alterations. We obtained the same phenotype upon introduction of RNAi constructs aimed at silencing the RAD51 gene at meiosis in dmc1 mutant plants. The meiotic figures we observed strongly suggest that homologous recombination is highly disturbed in these meiotic cells, leaving aberrant recombination events to repair the meiotic double-strand breaks. The 'brca2' meiotic phenotype was eliminated in spo11 mutant plants. Our experiments point to an essential role of Brca2 at meiosis in Arabidopsis. We also propose a role for Rad51 in the dmc1 context.     

Yeast Rad52 and Rad51 recombination proteins define a second pathway of DNA damage assessment in response to a single double-strand break

Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G(2)/M arrest. We have found that both Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51Delta. rad52Delta also suppresses adaptation defects of srs2Delta mutants but not those of yku70Delta or tid1Delta mutants. Neither rad54Delta nor rad55Delta affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Delta mutants and fully restores adaptation in yku70Delta and tid1Delta mutants. Surprisingly, although neither rfa1-t11 nor rad52Delta mutants are adaptation defective, the rad52Delta rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction. We suggest that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p.     

Recombination protein Tid1p controls resolution of cohesin-dependent linkages in meiosis in Saccharomyces cerevisiae

Sister chromatid cohesion and interhomologue recombination are coordinated to promote the segregation of homologous chromosomes instead of sister chromatids at the first meiotic division. During meiotic prophase in Saccharomyces cerevisiae, the meiosis-specific cohesin Rec8p localizes along chromosome axes and mediates most of the cohesion. The mitotic cohesin Mcd1p/Scc1p localizes to discrete spots along chromosome arms, and its function is not clear. In cells lacking Tid1p, which is a member of the SWI2/SNF2 family of helicase-like proteins that are involved in chromatin remodeling, Mcd1p and Rec8p persist abnormally through both meiotic divisions, and chromosome segregation fails in the majority of cells. Genetic results indicate that the primary defect in these cells is a failure to resolve Mcd1p-mediated connections. Tid1p interacts with recombination enzymes Dmc1p and Rad51p and has an established role in recombination repair. We propose that Tid1p remodels Mcd1p-mediated cohesion early in meiotic prophase to facilitate interhomologue recombination and the subsequent segregation of homologous chromosomes.     

RAD51B plays an essential role during somatic and meiotic recombination in Physcomitrella

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified in yeast (Rad55, Rad57 and Dmc1), plants and vertebrates (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1). RAD51 and DMC1 are the strand-exchange proteins forming a nucleofilament for strand invasion, however, the function of the paralogues in the process of homologous recombination is less clear. In yeast the two Rad51 paralogues, Rad55 and Rad57, have been shown to be involved in somatic and meiotic HR and they are essential to the formation of the Rad51/DNA nucleofilament counterbalancing the anti-recombinase activity of the SRS2 helicase. Here, we examined the role of RAD51B in the model bryophyte Physcomitrella patens. Mutant analysis shows that RAD51B is essential for the maintenance of genome integrity, for resistance to DNA damaging agents and for gene targeting. Furthermore, we set up methods to investigate meiosis in Physcomitrella and we demonstrate that the RAD51B protein is essential for meiotic homologous recombination. Finally, we show that all these functions are independent of the SRS2 anti-recombinase protein, which is in striking contrast to what is found in budding yeast where the RAD51 paralogues are fully dependent on the SRS2 anti-recombinase function.     

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.     

Stimulation of Dmc1-mediated DNA strand exchange by the human Rad54B protein

The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosis-specific Rad51 homolog. In the present study, we found that Rad54B enhanced the DNA strand-exchange activity of Dmc1 by stabilizing the Dmc1–single-stranded DNA (ssDNA) complex. Therefore, Rad54B may stimulate the Dmc1-mediated DNA strand exchange by stabilizing the nucleoprotein filament, which is formed on the ssDNA tails produced at DSB sites during homologous recombination.     

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.     

Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination

Homologous recombination (HR) is a primary DNA double-strand breaks (DSBs) repair mechanism. The recombinases Rad51 and Dmc1 are highly conserved in the RecA family; Rad51 is mainly responsible for DNA repair in somatic cells during mitosis while Dmc1 only works during meiosis in germ cells. This spatiotemporal difference is probably due to their distinctive mismatch tolerance during HR: Rad51 does not permit HR in the presence of mismatches, whereas Dmc1 can tolerate certain mismatches. Here, the cryo-EM structures of Rad51–DNA and Dmc1–DNA complexes revealed that the major conformational differences between these two proteins are located in their Loop2 regions, which contain invading single-stranded DNA (ssDNA) binding residues and double-stranded DNA (dsDNA) complementary strand binding residues, stabilizing ssDNA and dsDNA in presynaptic and postsynaptic complexes, respectively. By combining molecular dynamic simulation and single-molecule FRET assays, we identified that V273 and D274 in the Loop2 region of human RAD51 (hRAD51), corresponding to P274 and G275 of human DMC1 (hDMC1), are the key residues regulating mismatch tolerance during strand exchange in HR. This HR accuracy control mechanism provides mechanistic insights into the specific roles of Rad51 and Dmc1 in DNA double-strand break repair and may shed light on the regulatory mechanism of genetic recombination in mitosis and meiosis.     

DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression.

DMC1 is a new meiosis-specific yeast gene. Dmc1 protein is structurally similar to bacterial RecA proteins. dmc1 mutants are defective in reciprocal recombination, accumulate double-strand break (DSB) recombination intermediates, fail to form normal synaptonemal complex (SC), and arrest late in meiotic prophase. dmc1 phenotypes are consistent with a functional relationship between Dmc1 and RecA, and thus eukaryotic and prokaryotic mechanisms for homology recognition and strand exchange may be related. dmc1 phenotypes provide further evidence that recombination and SC formation are interrelated processes and are consistent with a requirement for DNA-DNA interactions during SC formation. dmc1 mutations confer prophase arrest. Additional evidence suggests that arrest occurs at a meiosis-specific cell cycle "checkpoint" in response to a primary defect in prophase chromosome metabolism. DMC1 is homologous to yeast's RAD51 gene, supporting the view that mitotic DSB repair has been recruited for use in meiotic chromosome metabolism.     

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.     

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.     

Tid1/Rdh54 translocase is phosphorylated through a Mec1- and Rad53-dependent manner in the presence of DSB lesions in budding yeast

Saccharomyces cerevisiae cells with a single double-strand break (DSB) activate the ATR/Mec1-dependent checkpoint response as a consequence of extensive ssDNA accumulation. The recombination factor Tid1/Rdh54, a member of the Swi2-like family proteins, has an ATPase activity and may contribute to the remodelling of nucleosomes on DNA. Tid1 dislocates Rad51 recombinase from dsDNA, can unwind and supercoil DNA filaments, and has been implicated in checkpoint adaptation from a G2/M arrest induced by an unrepaired DSB.Here we show that both ATR/Mec1 and Chk2/Rad53 kinases are implicated in the phosphorylation of Tid1 in the presence of DNA damage, indicating that the protein is regulated during the DNA damage response. We show that Tid1 ATPase activity is dispensable for its phosphorylation and for its recruitment near a DSB, but it is required to switch off Rad53 activation and for checkpoint adaptation. Mec1 and Rad53 kinases, together with Rad51 recombinase, are also implicated in the hyper-phosphorylation of the ATPase defective Tid1-K318R variant and in the efficient binding of the protein to the DSB site.In summary, Tid1 is a novel target of the DNA damage checkpoint pathway that is also involved in checkpoint adaptation.     

Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1: a molecular link between recombination and chromatin assembly during meiosis

In eukaryotes, meiosis leads to genetically variable gametes through recombination between homologous chromosomes of maternal and paternal origin. Chromatin organization following meiotic recombination is critical to ensure the correct segregation of homologous chromosomes into gametes. However, the mechanism of chromatin organization after meiotic recombination is unknown. In this study we report that the meiosis-specific recombinase Lim15/Dmc1 interacts with the homologue of the largest subunit of chromatin assembly factor 1 (CAF-1) in the basidiomycete Coprinopsis cinerea (Coprinus cinereus). Using C. cinerea LIM15/DMC1 (CcLIM15) as the bait in a yeast two-hybrid screen, we have isolated the C. cinerea homologue of Cac1, the largest subunit of CAF-1 in Saccharomyces cerevisiae, and named it C. cinerea Cac1-like (CcCac1L). Two-hybrid assays confirmed that CcCac1L binds CcLim15 in vivo. beta-Galactosidase assays revealed that the N-terminus of CcCac1L preferentially interacts with CcLim15. Co-immunoprecipitation experiments showed that these proteins also interact in the crude extract of meiotic cells. Furthermore, we demonstrate that, during meiosis, CcCac1L interacts with proliferating cell nuclear antigen (PCNA), a component of the DNA synthesis machinery recently reported as an interacting partner of Lim15/Dmc1. Taken together, these results suggest a novel role of the CAF-1-PCNA complex in meiotic events. We propose that the CAF-1-PCNA complex modulates chromatin assembly following meiotic recombination.     

AtPRD1 is required for meiotic double strand break formation in Arabidopsis thaliana

The initiation of meiotic recombination by the formation of DNA double-strand breaks (DSBs) catalysed by the Spo11 protein is strongly evolutionary conserved. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation, but, unlike Spo11, few of these proteins seem to be conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we have isolated a new gene, AtPRD1, whose mutation affects meiosis in Arabidopsis thaliana. In Atprd1 mutants, meiotic recombination rates fall dramatically, early recombination markers (e.g., DMC1 foci) are absent, but meiosis progresses until achiasmatic univalents are formed. Besides, Atprd1 mutants suppress DSB repair defects of a large range of meiotic mutants, showing that AtPRD1 is involved in meiotic recombination and is required for meiotic DSB formation. Furthermore, we showed that AtPRD1 and AtSPO11-1 interact in a yeast two-hybrid assay, suggesting that AtPRD1 could be a partner of AtSPO11-1. Moreover, our study reveals similarity between AtPRD1 and the mammalian protein Mei1, suggesting that AtPRD1 could be a Mei1 functional homologue.