A Dominant, Recombination-Defective Allele of Dmc1 Causing Male-Specific Sterility

DMC1 is a meiosis-specific homolog of bacterial RecA and eukaryotic RAD51 that can catalyze homologous DNA strand invasion and D-loop formation in vitro. DMC1-deficient mice and yeast are sterile due to defective meiotic recombination and chromosome synapsis. The authors identified a male dominant sterile allele of Dmc1, Dmc1^Mei11, encoding a missense mutation in the L2 DNA binding domain that abolishes strand invasion activity. Meiosis in male heterozygotes arrests in pachynema, characterized by incomplete chromosome synapsis and no crossing-over. Young heterozygous females have normal litter sizes despite having a decreased oocyte pool, a high incidence of meiosis I abnormalities, and susceptibility to premature ovarian failure. Dmc1^Mei11 exposes a sex difference in recombination in that a significant portion of female oocytes can compensate for DMC1 deficiency to undergo crossing-over and complete gametogenesis. Importantly, these data demonstrate that dominant alleles of meiosis genes can arise and propagate in populations, causing infertility and other reproductive consequences due to meiotic prophase I defects.     

Mouse pachytene checkpoint 2 (trip13) is required for completing meiotic recombination but not synapsis

In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice.     

MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination

Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB). This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 ^−/− spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob ^−/− meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.     

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.     

Mei1 is epistatic to Dmc1 during mouse meiosis

The Mei1^m1Jcs allele contains a point mutation in a novel gene required for normal meiosis in male and female mice. We previously hypothesized that Mei1 is likely required for the formation of genetically programmed double-strand breaks (DSBs), the initiating event of meiotic recombination because in mutant spermatocytes (1) RAD51 foci are greatly reduced at zygonema; (2) RAD51 foci can be restored by cisplatin-induced DNA damage; and (3) phosphorylated H2AX is greatly reduced at leptonema. If this hypothesis is correct, Mei1 would act upstream of genes required for repair of DSBs by homologous recombination. To test this, we examined meiosis in Mei^m1Jcs/Mei1^m1Jcs (Mei1^-/-) and Dmc1^tm1Jcs/Dmc1^tm1Jcs (Dmc1^-/-) mice and mice homozygous at both loci (Dmc1^-/- Mei1^-/-), exploiting the fact that oogenesis is much more severely affected by the absence of DMC1 than by the absence of MEI1. The phenotypes of both male and female double mutants were identical to that of Mei1^-/- animals. Therefore, Mei1 can be positioned upstream of Dmc1 in the genetic pathway that operates during mammalian meiosis. Furthermore, this epistatic interaction provides additional evidence in support of the hypothesis that Mei1 is required for the initiating events of meiotic recombination.     

The dual role of HOP2 in mammalian meiotic homologous recombination

Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2–MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1^−/− spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2–MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis.     

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.     

Meiotic Recombination in Arabidopsis Is Catalysed by DMC1, with RAD51 Playing a Supporting Role

Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis.     

Together yes, but not coupled: new insights into the roles of RAD51 and DMC1 in plant meiotic recombination

The eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, non-homologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.     

Multiple Pathways Suppress Non-Allelic Homologous Recombination during Meiosis in Saccharomyces cerevisiae

Recombination during meiosis in the form of crossover events promotes the segregation of homologous chromosomes by providing the only physical linkage between these chromosomes. Recombination occurs not only between allelic sites but also between non-allelic (ectopic) sites. Ectopic recombination is often suppressed to prevent non-productive linkages. In this study, we examined the effects of various mutations in genes involved in meiotic recombination on ectopic recombination during meiosis. RAD24, a DNA damage checkpoint clamp-loader gene, suppressed ectopic recombination in wild type in the same pathway as RAD51. In the absence of RAD24, a meiosis-specific recA homolog, DMC1, suppressed the recombination. Homology search and strand exchange in ectopic recombination occurred when either the RAD51 or the DMC1 recA homolog was absent, but was promoted by RAD52. Unexpectedly, the zip1 mutant, which is defective in chromosome synapsis, showed a decrease, rather than an increase, in ectopic recombination. Our results provide evidence for two types of ectopic recombination: one that occurs in wild-type cells and a second that occurs predominantly when the checkpoint pathway is inactivated.     

Matefin/SUN-1 Phosphorylation Is Part of a Surveillance Mechanism to Coordinate Chromosome Synapsis and Recombination with Meiotic Progression and Chromosome Movement

Faithful chromosome segregation during meiosis I depends on the establishment of a crossover between homologous chromosomes. This requires induction of DNA double-strand breaks (DSBs), alignment of homologs, homolog association by synapsis, and repair of DSBs via homologous recombination. The success of these events requires coordination between chromosomal events and meiotic progression. The conserved SUN/KASH nuclear envelope bridge establishes transient linkages between chromosome ends and cytoskeletal forces during meiosis. In Caenorhabditis elegans, this bridge is essential for bringing homologs together and preventing nonhomologous synapsis. Chromosome movement takes place during synapsis and recombination. Concomitant with the onset of chromosome movement, SUN-1 clusters at chromosome ends associated with the nuclear envelope, and it is phosphorylated in a chk-2- and plk-2-dependent manner. Identification of all SUN-1 phosphomodifications at its nuclear N terminus allowed us to address their role in prophase I. Failures in recombination and synapsis led to persistent phosphorylations, which are required to elicit a delay in progression. Unfinished meiotic tasks elicited sustained recruitment of PLK-2 to chromosome ends in a SUN-1 phosphorylation–dependent manner that is required for continued chromosome movement and characteristic of a zygotene arrest. Furthermore, SUN-1 phosphorylation supported efficient synapsis. We propose that signals emanating from a failure to successfully finish meiotic tasks are integrated at the nuclear periphery to regulate chromosome end–led movement and meiotic progression. The single unsynapsed X chromosome in male meiosis is precluded from inducing a progression delay, and we found it was devoid of a population of phosphorylated SUN-1. This suggests that SUN-1 phosphorylation is critical to delaying meiosis in response to perturbed synapsis. SUN-1 may be an integral part of a checkpoint system to monitor establishment of the obligate crossover, inducible only in leptotene/zygotene. Unrepaired DSBs and unsynapsed chromosomes maintain this checkpoint, but a crossover intermediate is necessary to shut it down.     

Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11

Spo11, a protein first identified in yeast, is thought to generate the chromosome breaks that initiate meiotic recombination. We now report that disruption of mouse Spo11 leads to severe gonadal abnormalities from defective meiosis. Spermatocytes suffer apoptotic death during early prophase; oocytes reach the diplotene/dictyate stage in nearly normal numbers, but most die soon after birth. Consistent with a conserved function in initiating meiotic recombination, Dmc1/Rad51 focus formation is abolished. Spo11(-/-) meiocytes also display homologous chromosome synapsis defects, similar to fungi but distinct from flies and nematodes. We propose that recombination initiation precedes and is required for normal synapsis in mammals. Our results also support the view that mammalian checkpoint responses to meiotic recombination and/or synapsis defects are sexually dimorphic.     

Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

Meiosis depends on homologous recombination (HR) in most sexually reproducing organisms. Efficient meiotic HR requires the activity of the meiosis-specific recombinase, Dmc1. Previous work shows Dmc1 is expressed in Entamoeba histolytica, a eukaryotic parasite responsible for amoebiasis throughout the world, suggesting this organism undergoes meiosis. Here, we demonstrate Dmc1 protein is expressed in E. histolytica. We show that purified ehDmc1 forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. The DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. In combination, calcium and Hop2-Mnd1 dramatically increase the rate of DNA strand exchange activity of ehDmc1. The biochemical system described herein provides a basis on which to better understand the role of ehDmc1 and other HR proteins in E. histolytica.     

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 Mei5-Sae3 Protein Complex Mediates Dmc1 Activity in Saccharomyces cerevisiae

During homologous recombination, a number of proteins cooperate to catalyze the loading of recombinases onto single-stranded DNA. Single-stranded DNA-binding proteins stimulate recombination by coating single-stranded DNA and keeping it free of secondary structure; however, in order for recombinases to load on single-stranded-DNA-binding protein-coated DNA, the activity of a class of proteins known as recombination mediators is required. Mediator proteins coordinate the handoff of single-stranded DNA from single-stranded DNA-binding protein to recombinase. Here we show that a complex of Mei5 and Sae3 from Saccharomyces cerevisiae preferentially binds single-stranded DNA and relieves the inhibition of the strand assimilation and DNA binding abilities of the meiotic recombinase Dmc1 imposed by the single-stranded DNA-binding protein replication protein A. Additionally, we demonstrate the physical interaction of Mei5-Sae3 with replication protein A. Our results, together with previous in vivo studies, indicate that Mei5-Sae3 is a mediator of Dmc1 assembly during meiotic recombination in S. cerevisiae.During meiosis, recombination between homologous chromosomes ensures proper segregation into haploid products. Recombination events are initiated by the formation of double strand breaks (DSBs)² in DNA (1). This is followed by resection of free DNA ends to yield 3′ single-stranded tails, upon which recombinase assembles to form nucleoprotein filaments. Following recombinase assembly, the nucleoprotein filament engages a donor chromatid, searches for homologous DNA sequences on that chromatid, and promotes strand exchange to yield a heteroduplex DNA intermediate often referred to as a joint molecule. Although recombinase alone is capable of promoting homology search and strand exchange in vitro, genetic and biochemical studies have demonstrated that normal recombinase function in vivo requires the activity of a number of accessory factors (2). These factors enhance the assembly of nucleoprotein filaments, target capture, homology search, and dissociation of recombinase from duplex DNA.Most eukaryotes possess two recombinases, both homologues of the Escherichia coli recombinase RecA: Rad51, which is the major recombinase in mitotic cells and is also important during meiotic recombination, and Dmc1, which functions only in meiosis. Dmc1 and Rad51 have been shown to assemble at DSBs by immunofluorescence and chromatin immunoprecipitation (3–6), and both proteins oligomerize on single-stranded DNA (ssDNA) to form nucleofilaments that catalyze strand invasion (7–9).A number of biochemical studies have defined the role of accessory factors in stimulating the activity of Rad51 (10–12). Replication protein A (RPA), the yeast ssDNA-binding protein (SSB), removes secondary structure in ssDNA that otherwise prevents formation of fully functional nucleoprotein filaments (13). Both Rad52 protein (11, 12) and the heterodimeric protein Rad55/Rad57 (14) can overcome the inhibitory effect of RPA on Rad51 nucleoprotein filament formation in purified systems, mediating a handoff between RPA and Rad51. It is thought that the mechanism for the mediator activity of Rad52 involves Rad52 recognizing and binding to RPA-coated ssDNA, where it provides nucleation sites for the recruitment of free molecules of Rad51 (15). The tumor suppressor protein BRCA2 also serves as an assembly factor for Rad51 during mitosis in a variety of species that encode orthologues of this protein, including mice (16), corn smut (17), and humans (18).The meiosis-specific recombinase Dmc1 is stimulated by a distinct set of accessory factors. Immunostaining studies suggest that the Rad51 mediators Rad52 and Rad55/Rad57 are not required for assembly of Dmc1 foci in vivo, although Rad51 itself promotes Dmc1 foci (19–21). More recently, immunostaining and chromatin immunoprecipitation experiments demonstrated a role for the Mei5 and Sae3 proteins of Saccharomyces cerevisiae in assembly of Dmc1 at sites of DSBs in vivo (22, 23). Consistent with these observations, mei5 and sae3 mutants display markedly similar meiotic defects as compared with dmc1 mutants, including defects in sporulation, spore viability, crossing over, DSB repair, progression through meiosis, and synaptonemal complex formation (19, 22–24). Finally, the three proteins have been shown to physically interact; Mei5 and Sae3 have been co-purified and co-immunoprecipitated, and an N-terminal portion of Mei5 has been shown to interact with Dmc1 in a two-hybrid assay (22).The fission yeast Schizosaccharomyces pombe encodes two proteins, Swi5 and Sfr1, which share sequence homology with Sae3 and Mei5, respectively (22). Swi5 and Sfr1 have been shown to stimulate the strand exchange activity of Rhp51 (the S. pombe Rad51 homologue) and Dmc1 (25). Although some results indicate functional similarity of Swi5-Sfr1 and Mei5-Sae3, there are also clear differences. The Mei5-Sae3 complex of budding yeast is expressed solely during meiosis, and no mitotic phenotypes have been reported for mei5 or sae3 mutants (22, 24, 26). In contrast, the Swi5-Sfr1 complex of fission yeast is expressed in mitotic and meiotic cells, and mutations in SWI5 have been shown to cause defects in mitotic recombination (27). Furthermore, although mei5 and sae3 mutants are phenotypically similar to dmc1 mutants, swi5 and sfr1 mutants display more severe meiotic defects during fission yeast meiosis than do dmc1 mutants (27–29). These data suggest that although Swi5-Sfr1 clearly contributes to Rad51 activity in fission yeast, it is possible that the activity of Mei5-Sae3 is restricted to stimulating Dmc1 in budding yeast.In this study, a biochemical approach is used to test the budding yeast Mei5-Sae3 complex for properties expected of a recombinase assembly mediator. We show that Mei5-Sae3 binds both ssDNA and double-stranded DNA (dsDNA) but binds ssDNA preferentially. We also show that Mei5-Sae3 can overcome the inhibitory effects of RPA on the ssDNA binding and strand assimilation activities of Dmc1. Finally, we show that Mei5-Sae3 and RPA bind one another directly. These results indicate that Mei5-Sae3 acts directly as a mediator protein for assembly of Dmc1.     

AtMND1 is required for homologous pairing during meiosis in Arabidopsis

Background  

Pairing of homologous chromosomes at meiosis is an important requirement for recombination and balanced chromosome segregation among the products of meiotic division. Recombination is initiated by double strand breaks (DSBs) made by Spo11 followed by interaction of DSB sites with a homologous chromosome. This interaction requires the strand exchange proteins Rad51 and Dmc1 that bind to single stranded regions created by resection of ends at the site of DSBs and promote interactions with uncut DNA on the homologous partner. Recombination is also considered to be dependent on factors that stabilize interactions between homologous chromosomes. In budding yeast Hop2 and Mnd1 act as a complex to promote homologous pairing and recombination in conjunction with Rad51 and Dmc1.     

Hop2-Mnd1 condenses DNA to stimulate the synapsis phase of DNA strand exchange

Hop2-Mnd1 is a meiotic recombination mediator that stimulates DNA strand invasion by both Dmc1 and Rad51. To understand the biochemical mechanism of this stimulation, we directly visualized the heterodimer acting on single molecules of duplex DNA using optical tweezers and video fluorescence microscopy. The results show that the Hop2-Mnd1 heterodimer efficiently condenses double-stranded DNA via formation of a bright spot or DNA condensate. The condensation of DNA is Hop2-Mnd1 concentration-dependent, reversible, and specific to the heterodimer, as neither Hop2 nor Mnd1 acting alone can facilitate this reaction. The results also show that the rate-limiting nucleation step of DNA condensation is overcome in the presence of divalent metal ions, with the following order of preference: Mn²⁺>Mg²⁺>Ca²⁺. Hop2-Mnd1/Dmc1/single-stranded DNA nucleoprotein filaments also condense double-stranded DNA in a heterodimer concentration-dependent manner. Of importance, the concentration dependence parallels that seen in DNA strand exchange. We propose that rapid DNA condensation is a key factor in stimulating synapsis, whereas decondensation may facilitate the invasion step and/or the ensuing branch migration process.     

Improper chromosome synapsis is associated with elongated RAD51 structures in the maize<Emphasis Type="Italic"> desynaptic2</Emphasis> mutant

The RecA homolog, RAD51, performs a central role in catalyzing the DNA strand exchange event of meiotic recombination. During meiosis, RAD51 complexes develop on pairing chromosomes and then most disappear upon synapsis. In the maize meiotic mutant desynaptic2 (dsy2), homologous chromosome pairing and recombination are reduced by ~70% in male meiosis. Fluorescent in situ hybridization studies demonstrate that a normal telomere bouquet develops but the pairing of a representative gene locus is still only 25%. Chromosome synapsis is aberrant as exemplified by unsynapsed regions of the chromosomes. In the mutant, we observed unusual RAD51 structures during chromosome pairing. Instead of spherical single and double RAD51 structures, we saw long thin filaments that extended along or around a single chromosome or stretched between two widely separated chromosomes. Mapping with simple sequence repeat (SSR) markers places the dsy2 gene to near the centromere on chromosome 5, therefore it is not an allele of rad51. Thus, the normal dsy2 gene product is required for both homologous chromosome synapsis and proper RAD51 filament behavior when chromosomes pair. Edited by: P. Moens     

DMC1 attenuates RAD51-mediated recombination in Arabidopsis

Ensuring balanced distribution of chromosomes in gametes, meiotic recombination is essential for fertility in most sexually reproducing organisms. The repair of the programmed DNA double strand breaks that initiate meiotic recombination requires two DNA strand-exchange proteins, RAD51 and DMC1, to search for and invade an intact DNA molecule on the homologous chromosome. DMC1 is meiosis-specific, while RAD51 is essential for both mitotic and meiotic homologous recombination. DMC1 is the main catalytically active strand-exchange protein during meiosis, while this activity of RAD51 is downregulated. RAD51 is however an essential cofactor in meiosis, supporting the function of DMC1. This work presents a study of the mechanism(s) involved in this and our results point to DMC1 being, at least, a major actor in the meiotic suppression of the RAD51 strand-exchange activity in plants. Ectopic expression of DMC1 in somatic cells renders plants hypersensitive to DNA damage and specifically impairs RAD51-dependent homologous recombination. DNA damage-induced RAD51 focus formation in somatic cells is not however suppressed by ectopic expression of DMC1. Interestingly, DMC1 also forms damage-induced foci in these cells and we further show that the ability of DMC1 to prevent RAD51-mediated recombination is associated with local assembly of DMC1 at DNA breaks. In support of our hypothesis, expression of a dominant negative DMC1 protein in meiosis impairs RAD51-mediated DSB repair. We propose that DMC1 acts to prevent RAD51-mediated recombination in Arabidopsis and that this down-regulation requires local assembly of DMC1 nucleofilaments.     

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.