Interactions between Mei4, Rec114, and other proteins required for meiotic DNA double-strand break formation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In most sexually reproducing organisms, meiotic recombination is initiated by DNA double-strand breaks (DSBs) formed by the Spo11 protein. In budding yeast, nine other proteins are also required for DSB formation, but the roles of these proteins and the interactions among them are poorly understood. We report further studies of the behaviors of these proteins. Consistent with other studies, we find that Mei4 and Rec114 bind to chromosomes from leptonema through early pachynema. Both proteins showed only limited colocalization with the meiotic cohesin subunit Rec8, suggesting that Mei4 and Rec114 associated preferentially with chromatin loops. Rec114 localization was independent of other DSB factors, but Mei4 localization was strongly dependent on Rec114 and Mer2. Systematic deletion analysis identified protein regions important for a previously described two-hybrid interaction between Mei4 and Rec114. We also report functional characterization of a previously misannotated 5′ coding exon of REC102. Sequences encoded in this exon are essential for DSB formation and for Rec102 interaction with Rec104, Spo11, Rec114, and Mei4. Finally, we also examined genetic requirements for a set of previously described two-hybrid interactions that can be detected only when the reporter strain is induced to enter meiosis. This analysis reveals new functional dependencies for interactions among the DSB proteins. Taken together, these studies support the view that Mei4, Rec114, and Mer2 make up a functional subgroup that is distinct from other subgroups of the DSB proteins: Spo11–Ski8, Rec102–Rec104, and Mre11–Rad50–Xrs2. These studies also suggest that an essential function of Rec102 and Rec104 is to connect Mei4 and Rec114 to Spo11. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination

Meiotic recombination between homologous chromosomes initiates via programmed DNA double-strand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2, and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in?a domain-specific fashion by cohesin. Loss of Rec114, Mer2, and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.     

Functional interactions between SPO11 and REC102 during initiation of meiotic recombination in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, formation of the DNA double-strand breaks (DSBs) that initiate meiotic recombination requires the products of at least 10 genes. Spo11p is thought to be the catalytic subunit of the DNA cleaving activity, but the roles of the other proteins, and the interactions among them, are not well understood. This study demonstrates genetic and physical interactions between the products of SPO11 and another early meiotic gene required for DSB formation, REC102. We found that epitope-tagged versions of SPO11 and REC102 that by themselves were capable of supporting normal or nearly normal levels of meiotic recombination conferred a severe synthetic cold-sensitive phenotype when combined in the same cells. DSB formation, meiotic gene conversion, and spore viability were drastically reduced in the doubly tagged strain at a nonpermissive temperature. This conditional defect could be partially rescued by expression of untagged SPO11, but not by expression of untagged REC102, indicating that tagged REC102 is fully dominant for this synthetic phenotype. Both tagged and wild-type Spo11p co-immunoprecipitated with tagged Rec102p from meiotic cell extracts, indicating that these proteins are present in a common complex in vivo. Tagged Rec102p localized to the nucleus in whole cells and to chromatin on spread meiotic chromosomes. Our results are consistent with the idea that a multiprotein complex that includes Spo11p and Rec102p promotes meiotic DSB formation.     

Spatial organization and dynamics of the association of Rec102 and Rec104 with meiotic chromosomes

Meiotic double-strand breaks (DSBs) are formed by Spo11 in conjunction with at least nine other proteins whose roles are not well understood. We find that two of these proteins, Rec102 and Rec104, interact physically, are mutually dependent for proper subcellular localization, and share a requirement for Spo11 and Ski8 for their recruitment to meiotic chromosomes, suggesting that they work together as a functional unit. Rec102 associated extensively with chromatin loops during leptotene and zygotene and showed preferential binding in the vicinity at least of most DSB sites, consistent with a direct role in DSB formation. However, Rec102 was associated with both DSB-hot and DSB-cold regions, ruling out a simple model in which sites of DSB formation are dictated by where Rec102/104 complexes load. Both proteins persisted on chromatin until pachytene before abruptly disappearing, indicating that they remain on chromosomes well after DSB formation. These studies reveal unexpected behaviors for Rec102 and Rec104, and point to distinct roles and subcomplexes among the DSB proteins.     

The Mre11 complex influences DNA repair, synapsis, and crossing over in murine meiosis

The Mre11 complex (consisting of MRE11, RAD50, and NBS1/Xrs2) is required for double-strand break (DSB) formation, processing, and checkpoint signaling during meiotic cell division in S. cerevisiae. Whereas studies of Mre11 complex mutants in S. pombe and A. thaliana indicate that the complex has other essential meiotic roles , relatively little is known regarding the functions of the complex downstream of meiotic break formation and processing or its role in meiosis in higher eukaryotes. We analyzed meiotic events in mice harboring hypomorphic Mre11 and Nbs1 mutations which, unlike null mutants, support viability . Our studies revealed defects in the temporal progression of meiotic prophase, incomplete and aberrant synapsis of homologous chromosomes, persistence of strand exchange proteins, and alterations in both the frequency and placement of MLH1 foci, a marker of crossovers. A unique sex-dependent effect on MLH1 foci and chiasmata numbers was observed: males exhibited an increase and females a decrease in recombination levels. Thus, our findings implicate the Mre11 complex in meiotic DNA repair and synapsis in mammals and indicate that the complex may contribute to the establishment of normal sex-specific differences in meiosis.     

Conservation and divergence of meiotic DNA double strand break forming mechanisms in Arabidopsis thaliana

In the current meiotic recombination initiation model, the SPO11 catalytic subunits associate with MTOPVIB to form a Topoisomerase VI-like complex that generates DNA double strand breaks (DSBs). Four additional proteins, PRD1/AtMEI1, PRD2/AtMEI4, PRD3/AtMER2 and the plant specific DFO are required for meiotic DSB formation. Here we show that (i) MTOPVIB and PRD1 provide the link between the catalytic sub-complex and the other DSB proteins, (ii) PRD3/AtMER2, while localized to the axis, does not assemble a canonical pre-DSB complex but establishes a direct link between the DSB-forming and resection machineries, (iii) DFO controls MTOPVIB foci formation and is part of a divergent RMM-like complex including PHS1/AtREC114 and PRD2/AtMEI4 but not PRD3/AtMER2, (iv) PHS1/AtREC114 is absolutely unnecessary for DSB formation despite having a conserved position within the DSB protein network and (v) MTOPVIB and PRD2/AtMEI4 interact directly with chromosome axis proteins to anchor the meiotic DSB machinery to the axis.     

Association of Mre11p with double-strand break sites during yeast meiosis

The repair of DNA double-strand breaks (DSBs) requires the activity of the Mre11/Rad50/Xrs2(Nbs1) complex. In Saccharomyces cerevisiae, this complex is required for both the initiation of meiotic recombination by Spo11p-catalyzed programmed DSBs and for break end resection, which is necessary for repair by homologous recombination. We report that Mre11p transiently associates with the chromatin of Spo11-dependent DSB regions throughout the genome. Mutant analyses show that Mre11p binding requires the function of all genes required for DSB formation, with the exception of RAD50. However, Mre11p binding does not require DSB formation itself, since Mre11p transiently associates with DSB regions in the catalysis-negative mutant spo11-Y135F. Mre11p release from chromatin is blocked in mutants that accumulate unresected DSBs. We propose that Mre11p is a component of a pre-DSB complex that assembles on the DSB sites, thus ensuring a tight coupling between DSB formation by Spo11p and the processing of break ends.     

DNA damage-dependent nuclear dynamics of the Mre11 complex

The Mre11 complex has been implicated in diverse aspects of the cellular response to DNA damage. We used in situ fractionation of human fibroblasts to carry out cytologic analysis of Mre11 complex proteins in the double-strand break (DSB) response. In situ fractionation removes most nucleoplasmic protein, permitting immunofluorescent localization of proteins that become more avidly bound to nuclear structures after induction of DNA damage. We found that a fraction of the Mre11 complex was bound to promyelocyte leukemia protein bodies in undamaged cells. Within 10 min after gamma irradiation, nuclear retention of the Mre11 complex in small granular foci was observed and persisted until 2 h postirradiation. In light of the previous demonstration that the Mre11 complex associated with ionizing radiation (IR)-induced DSBs, we infer that the protein retained under these conditions was associated with DNA damage. We also observed increased retention of Rad51 following IR treatment, although IR induced Rad51 foci were distinct from Mre11 foci. The ATM kinase, which phosphorylates Nbs1 during activation of the S-phase checkpoint, was not required for the Mre11 complex to associate with DNA damage. These data suggest that the functions of the Mre11 complex in the DSB response are implicitly dependent upon its ability to detect DNA damage.     

Sae2p phosphorylation is crucial for cooperation with Mre11p for resection of DNA double-strand break ends during meiotic recombination in Saccharomyces cerevisiae

Meiotic recombination is initiated by the introduction of DNA double-strand breaks (DSBs) at recombination hotspots. DSB ends are resected to yield ssDNA, which is used in a homology search. Sae2p, which is involved in the resection of DSB ends, is phosphorylated by the Mec1p and Tel1p kinases during meiosis. To clarify the role of Sae2p phosphorylation in meiotic recombination, three mutants with alanine substitutions (at two putative Mec1/Tel1 phosphorylation sites near the N terminus, at three sites near the C terminus or at all five sites) were constructed. Analysis of DSB ends during meiotic recombination demonstrated that phosphorylation of the three C-terminal phosphorylation sites is necessary for DSB end resection and that phosphorylation of the two N-terminal phosphorylation sites is required for the efficient initiation of DSB end resection. Sae2p was localized on meiotic chromosomes in the rad50S and mre11-H125R mutants, which accumulate DSB ends. Alanine substitutions of all phosphorylation sites did not affect localization of Sae2p on meiotic chromosomes. Although colocalization of Sae2p with Mre11p and recombinant formation were observed in the N-terminally mutated and the C-terminally mutated strains, these processes were drastically impaired in the quintuple mutant. These results indicate that phosphorylation of Sae2p is required to initiate resection and to improve the efficiency of resection through cooperation with the Mre11-Rad50-Xrs2 complex.     

Meiotic recombination proteins localize to linear elements in Schizosaccharomyces pombe

In fission yeast, meiotic prophase nuclei develop structures known as linear elements (LinEs), instead of a canonical synaptonemal complex. LinEs contain Rec10 protein. While Rec10 is essential for meiotic recombination, the precise role of LinEs in this process is unknown. Using in situ immunostaining, we show that Rec7 (which is required for meiosis-specific DNA double-strand break (DSB) formation) aggregates in foci on LinEs. The strand exchange protein Rad51, which is known to mark the sites of DSBs, also localizes to LinEs, although to a lesser degree. The number of Rec7 foci corresponds well with the average number of genetic recombination events per meiosis suggesting that Rec7 marks the sites of recombination. Rec7 and Rad51 foci do not co-localize, presumably because they act sequentially on recombination sites. The localization of Rec7 is dependent on Rec10 but independent of the DSB-inducing protein Rec12/Spo11. Neither Rec7 nor Rad51 localization depends on the LinE-associated proteins Hop1 and Mek1, but the formation of Rad51 foci depends on Rec10, Rec7, and, as expected, Rec12/Spo11. We propose that LinEs form around designated recombination sites before the induction of DSBs and that most, if not all, meiotic recombination initiates within the setting provided by LinEs.     

Arsenic-induced Mre11 phosphorylation is cell cycle-dependent and defective in NBS cells

Cancer-prone diseases ataxia-telangiectasia (AT), Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD) are defective in the repair of DNA double-stranded break (DSB). On the other hand, arsenic (As) has been reported to cause DSB and to be involved in the occurrence of skin, lung and bladder cancers. To dissect the repair mechanism of As-induced DSB, wild type, AT and NBS cells were treated with sodium arsenite to study the complex formation and post-translational modification of Rad50/NBS1/Mre11 repair proteins. Our results showed that Mre11 went through cell cycle-dependent phosphorylation upon sodium arsenite treatment and this post-translational modification required NBS1 but not ATM. Defective As-induced Mre11 phosphorylation was rescued by reconstitution with full length NBS1 in NBS cells. Although As-induced Mre11 phosphorylation was not required for Rad50/NBS1/Mre11 complex formation, it might be required for the formation of Rad50/NBS1/Mre11 nuclear foci upon DNA damage.     

mei-P22 encodes a chromosome-associated protein required for the initiation of meiotic recombination in Drosophila melanogaster

Double-strand breaks (DSB) initiate meiotic recombination in a variety of organisms. Here we present genetic evidence that the mei-P22 gene is required for the induction of DSBs during meiotic prophase in Drosophila females. Strong mei-P22 mutations eliminate meiotic crossing over and suppress the sterility of DSB repair-defective mutants. Interestingly, crossing over in mei-P22 mutants can be restored to almost 50% of wild-type by X irradiation. In addition, an antibody-based assay was used to demonstrate that DSBs are not formed in mei-P22 mutants. This array of phenotypes is identical to that of mei-W68 mutants; mei-W68 encodes the Drosophila Spo11 homolog that is proposed to be an enzyme required for DSB formation. Consistent with a direct role in DSB formation, mei-P22 encodes a basic 35.7-kD protein, which, when examined by immunofluorescence, localizes to foci on meiotic chromosomes. MEI-P22 foci appear transiently in early meiotic prophase, which is when meiotic recombination is believed to initiate. By using an antibody to C(3)G as a marker for synaptonemal complex (SC) formation, we observed that SC is present before MEI-P22 associates with the chromosomes, thus providing direct evidence that the development of SC precedes the initiation of meiotic recombination. Similarly, we found that MEI-P22 foci did not appear in a c(3)G mutant in which SC does not form, suggesting that DSB formation is dependent on SC formation in Drosophila. We propose that MEI-P22 interacts with meiosis-specific chromosome proteins to facilitate DSB creation by MEI-W68.     

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.     

Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination.

In Saccharomyces cerevisiae, Mre11 protein is involved in both double-strand DNA break (DSB) repair and meiotic DSB formation. Here, we report the correlation of nuclease and DNA-binding activities of Mre11 with its functions in DNA repair and meiotic DSB formation. Purified Mre11 bound to DNA efficiently and was shown to have Mn2+-dependent nuclease activities. A point mutation in the N-terminal phosphoesterase motif (Mre11D16A) resulted in the abolition of nuclease activities but had no significant effect on DNA binding. The wild-type level of nuclease activity was detected in a C-terminal truncated protein (Mre11DeltaC49), although it had reduced DNA-binding activity. Phenotypes of the corresponding mutations were also analyzed. The mre11D16A mutation conferred methyl methanesulfonate-sensitivity to mitotic cells and caused the accumulation of unprocessed meiotic DSBs. The mre11DeltaC49 mutant exhibited almost wild-type phenotypes in mitosis. However, in meiosis, no DSB formation could be detected and an aberrant chromatin configuration was observed at DSB sites in the mre11DeltaC49 mutant. These results indicate that Mre11 has two separable functional domains: the N-terminal nuclease domain required for DSB repair, and the C-terminal dsDNA-binding domain essential to its meiotic functions such as chromatin modification and DSB formation. Keywords: DNA binding/double-strand break repair/DSB formation/Mre11/nuclease     

Meiotic association between Spo11 regulated by Rec102, Rec104 and Rec114

Meiotic recombination is initiated by DNA double-stranded break (DSB) formation catalyzed by Spo11, a type-II topoisomerase-like transesterificase, presumably via a dimerization-mediated mechanism. We demonstrate the existence of in vivo interactions between Spo11 proteins carrying distinct tags, and the chromatin-binding and DSB activity of tagged Spo11at innate and targeted DSB sites upon fusion to the Gal4 DNA-binding domain. First we identified the interaction between Spo11-3FLAG and Gal4BD-Spo11 proteins, and established that this interaction specifically occurs at the time of DSB formation. We then observed that presence of the Gal4BD-spo11Y135F (nuclease-deficient) protein allows Spo11-3FLAG recruitment at the GAL2 locus, indicative of the formation of a hetero-complex near the GAL2 UAS sites, but no formation of double- or single-strand breaks. Spo11 self-interaction around the GAL2 DSB site depends on other proteins for DSB formation, in particular Rec102, Rec104 and Rec114. Together, these results suggest that in vivo self-association of Spo11 during meiosis is genetically regulated. The results are discussed in relation to possible roles of Spo11 self-interaction in the control of the cleavage activity.     

MRE11 and COM1/SAE2 are required for double-strand break repair and efficient chromosome pairing during meiosis of the protist Tetrahymena

Programmed DNA double-strand breaks (DSBs) are generated during meiosis to initiate homologous recombination. Various aspects of DSB formation, signaling, and repair are accomplished or governed by Mre11, a component of the MRN/MRX complex, partially in cooperation with Com1/Sae2/CtIP. We used Tetrahymena to study evolutionarily conserved and changed functions of Mre11 and Com1. There is a difference between organisms with respect to the dependency of meiotic DSB formation on Mre11. By cytology and an electrophoresis-based assay for DSBs, we found that in Tetrahymena Mre11p is not required for the formation and ATR-dependent signaling of DSBs. Its dispensability is also reflected by wild-type-like DSB-dependent reorganization of the meiotic nucleus and by the phosphorylation of H2A.X in mre11∆ mutant. However, mre11∆ and com1∆ mutants are unable to repair DSBs, and chromosome pairing is reduced. It is concluded that, while MRE11 has no universal role in DNA damage signaling, its requirement for DSB repair is conserved between evolutionarily distant organisms. Moreover, reduced chromosome pairing in repair-deficient mutants reveals the existence of two complementing pairing processes, one by the rough parallel arrangement of chromosomes imposed by the tubular shape of the meiotic nucleus and the other by repair-dependent precise sequence matching.     

MRE11 is required for homologous synapsis and DSB processing in rice meiosis

Mre11, a conserved protein found in organisms ranging from yeast to multicellular organisms, is required for normal meiotic recombination. Mre11 interacts with Rad50 and Nbs1/Xrs2 to form a complex (MRN/X) that participates in double-strand break (DSB) ends processing. In this study, we silenced the MRE11 gene in rice and detailed its function using molecular and cytological methods. The OsMRE11-deficient plants exhibited normal vegetative growth but could not set seed. Cytological analysis indicated that in the OsMRE11-deficient plants, homologous pairing was totally inhibited, and the chromosomes were completely entangled as a formation of multivalents at metaphase I, leading to the consequence of serious chromosome fragmentation during anaphase I. Immunofluorescence studies further demonstrated that OsMRE11 is required for homologous synapsis and DSB processing but is dispensable for meiotic DSB formation. We found that OsMRE11 protein was located on meiotic chromosomes from interphase to late pachytene. This protein showed normal localization in zep1, Oscom1 and Osmer3, as well as in OsSPO11-1 ^RNAi plants, but not in pair2 and pair3 mutants. Taken together, our results provide evidence that OsMRE11 performs a function essential for maintaining the normal HR process and inhibiting non-homologous recombination during meiosis.     

Meiotic localization of Mre11 and Rad50 in wild type, <Emphasis Type="Italic">spo11-1</Emphasis>, and MRN complex mutants of <Emphasis Type="Italic">Coprinus cinereus</Emphasis>

The Mre11-Rad50-Nbs1 (MRN) complex is required for numerous cellular processes that involve interactions with DNA double-strand breaks. For the majority of these processes, the MRN complex is thought to act as a unit, with each protein aiding the activity of the others. We have examined the relationship between Mre11 and Rad50 during meiosis in the basidiomycete Coprinus cinereus (Coprinopsis cinerea), investigating to what extent activities of Mre11 and Rad50 are interdependent. We showed that mre11-1 is epistatic to rad50-1 with respect to the time of meiotic arrest, indicating that Mre11 activity facilitates the diffuse diplotene arrest of rad50 mutants. Anti-Mre11 and anti-Rad50 antibodies were used to examine MRN complex localization in a wild-type strain and in spo11, mre11, and rad50 mutants. In wild type, numbers of Mre11 and Rad50 foci peaked at time points corresponding to leptotene and early zygotene. In the spo11-1 mutant, which is defective in meiotic double-strand break formation, foci accumulated throughout prophase I. Of seven MRN mutants examined, only two rad50 strains exhibited Mre11 and Rad50 foci that localized to chromatin, although Mre11 protein was found in the cell for all of them. Analysis of predicted mutant structures showed that stable localization of Mre11 and Rad50 does not depend upon a wild-type hook-proximal coiled coil, but does require the presence of the Rad50 ATPase/adenylate cyclase domains. We found that Mre11 and Rad50 were interdependent for binding to meiotic chromosomes. However, the majority of foci observed apparently contained only one of the two proteins. Independent Mre11 and Rad50 foci might indicate disassociation of the complex during meiosis or could reflect independent structural roles for the two proteins in meiotic chromatin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Support for a meiotic recombination initiation complex: interactions among Rec102p,Rec104p,and Spo11p

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