Meiotic defects in the<Emphasis Type="Italic"> Arabidopsis</Emphasis><Emphasis Type="Italic"> rad50</Emphasis> mutant point to conservation of the MRX complex function in early stages of meiotic recombination

The Rad50, Mre11 and Xrs2/Nbs1 proteins, which form the highly conserved MRX complex, perform a wide range of functions concerning the maintenance and function of DNA in eukaryotes. These include recombination, DNA repair, replication, telomere homeostasis and meiosis. Notwithstanding the attention paid to this complex, the inviability of vertebrate rad50 and mre11 mutants has led to a relative lack of information concerning the role of these proteins in meiosis in higher eukaryotes. We have previously reported that Arabidopsis atrad50 mutant plants are viable and that atrad50 mutant plants are sterile. The present study reports an analysis of the causes of this sterility and the implication of the AtRad50 protein in meiosis. Both male and female gametogenesis are defective in the Arabidopsis atrad50 mutant and cytological observation of male meiosis indicates that in the absence of the AtRad50 protein, homologous chromosomes are unable to synapse. Finally, the atrad50 mutation leads to the destruction of chromosomes during meiosis. These phenotypes support a role for the Arabidopsis MRX complex in early stages of meiotic recombination.     

Coprinus cinereus rad50 mutants reveal an essential structural role for Rad50 in axial element and synaptonemal complex formation, homolog pairing and meiotic recombination

The Mre11/Rad50/Nbs1 (MRN) complex is required for eukaryotic DNA double-strand break (DSB) repair and meiotic recombination. We cloned the Coprinus cinereus rad50 gene and showed that it corresponds to the complementation group previously named rad12, identified mutations in 15 rad50 alleles, and mapped two of the mutations onto molecular models of Rad50 structure. We found that C. cinereus rad50 and mre11 mutants arrest in meiosis and that this arrest is Spo11 dependent. In addition, some rad50 alleles form inducible, Spo11-dependent Rad51 foci and therefore must be forming meiotic DSBs. Thus, we think it likely that arrest in both mre11-1 and the collection of rad50 mutants is the result of unrepaired or improperly processed DSBs in the genome and that Rad50 and Mre11 are dispensable in C. cinereus for DSB formation, but required for appropriate DSB processing. We found that the ability of rad50 mutant strains to form Rad51 foci correlates with their ability to promote synaptonemal complex formation and with levels of stable meiotic pairing and that partial pairing, recombination initiation, and synapsis occur in the absence of wild-type Rad50 catalytic domains. Examination of single- and double-mutant strains showed that a spo11 mutation that prevents DSB formation enhances axial element (AE) formation for rad50-4, an allele predicted to encode a protein with intact hook region and hook-proximal coiled coils, but not for rad50-1, an allele predicted to encode a severely truncated protein, or for rad50-5, which encodes a protein whose hook-proximal coiled-coil region is disrupted. Therefore, Rad50 has an essential structural role in the formation of AEs, separate from the DSB-processing activity of the MRN complex.     

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

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

Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11-dependent genome fragmentation during meiosis

The Mre11/Rad50/Nbs1 complex is involved in many aspects of chromosome metabolism. Aberrant function of the complex is associated with defects in the DNA checkpoint, double-strand break repair, meiosis, and telomere maintenance. In this article, we report the consequences of Mre11 dysfunction for the stability of mitotic and meiotic chromosomes in Arabidopsis thaliana. Although plants homozygous for a T-DNA insertion in a conserved region of the MRE11 gene are viable, they exhibit growth defects and are infertile. Analysis of mitotic chromosomes prepared from the mutant plants revealed abundant dicentric chromosomes and chromosomal fragments. Fluorescence in situ hybridization showed that anaphase bridges are often formed by homologous chromosome arms. The frequency of chromosome fusions was not reduced in mre11 ku70 double mutants, suggesting that plants possess DNA end-joining activities independent of the Ku70/80 and Mre11 complexes. Cytogenetic examination of pollen mother cells revealed massive chromosome fragmentation and the absence of synapsis in the initial stages of meiosis. The fragmentation was substantially suppressed in mre11 spo11-1 double mutants, indicating that Mre11 is required for repair but not for the induction of Spo11-dependent meiotic DNA breaks in Arabidopsis.     

Characterization of the gene <Emphasis Type="Italic">Mre11</Emphasis> and evidence of silencing after polyploidization in <Emphasis Type="Italic">Triticum</Emphasis>

The MRE11 protein is a component of the highly conserved MRN complex, along with RAD50 and NBS1. This complex is crucial in the repair of breaks in double stranded DNA, and is involved in many other cell processes. The present paper reports the molecular characterization of Mre11 gene in all three genomes of wheat, making use of the diploid species Triticum monococcum (genome A) and Aegilops Tauschii (genome D), the tetraploid T. turgidum (genomes A and B), and the hexaploid T. aestivum (genomes A, B and D). The genomic sequences characterized ranged from 4,662 to 4,766 bp in length; the cDNA corresponding to the processed mRNA was 2,440–2,510 bp long. In all cases, Mre11 coded for a highly conserved protein of 699 amino acids with a structure involving 22 exons. Mre11 expression was determined by real-time PCR in all the species analysed. The tetraploid species showed an expression similar to that of the diploid Ae. tauschii and lower than that of T. monococcum. Stronger expression was detected in the hexaploid T. aestivum. The SSCP technique was modified by introducing fluorescent labelling to the procedure in order to analyse the expression of the different Mre11 genes (i.e., those belonging to the different genomes) in the polyploid species. In both polyploids, the Mre11 gene belonging to the B genome was the least expressed. This probably reflects a first step in the process of silencing duplicate genes after polyploidization.     

Distinct Roles of the ATR Kinase and the Mre11-Rad50-Nbs1 Complex in the Maintenance of Chromosomal Stability in Arabidopsis

Signaling of chromosomal DNA breaks is of primary importance for initiation of repair and, thus, for global genomic stability. Although the Mre11-Rad50-Nbs1 (MRN) complex is the first sensor of double-strand breaks, its role in double-strand break (DSB) signaling is not fully understood. We report the absence of γ-ray–induced, ATM/ATR-dependent histone H2AX phosphorylation in Arabidopsis thaliana rad50 and mre11 mutants, confirming that the MRN complex is required for H2AX phosphorylation by the ATM and ATR kinases in response to irradiation-induced DSB in Arabidopsis. rad50 and mre11 mutants spontaneously activate a DNA damage response, as shown by the presence of γ-H2AX foci and activation of cell cycle arrest in nonirradiated plants. This response is ATR dependent as shown both by the absence of these spontaneous foci and by the wild-type mitotic indices of double rad50 atr and mre11 atr plants. EdU S-phase labeling and fluorescence in situ hybridization analysis using specific subtelomeric probes point to a replicative S-phase origin of this chromosome damage in the double mutants and not to telomere destabilization. Thus, the data presented here show the exclusive involvement of ATR in DNA damage signaling in MRN mutants and provide evidence for a role for ATR in the avoidance of S-phase DNA damage.     

Effect of <Emphasis Type="Italic">rad50</Emphasis> mutation on illegitimate recombination in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genes in the RAD52 epistasis group are involved in repairing DNA double-stranded breaks via homologous recombination. We have previously shown that RAD50 is involved in mitotic nonhomologous integration but not in homologous integration. However, the role of Rad50 in nonhomologous integration has not previously been examined. In the current work, we report that the rad50∆ mutation caused a tenfold decrease in the frequency of nonhomologous integration with the majority of nonhomologous integrants showing an unstable Ura⁺ phenotype. Sequencing analysis of the integration target sites showed that integration events of both ends of the integrating vector in the rad50∆ mutant occurred at different chromosomal locations, resulting in large deletions or translocations on the genomic insertion sites. Interestingly, 47% of events in the rad50∆ mutant were integrated into repetitive sequences including rDNA locus, telomeres and Ty elements and 27% of events were integrated into non-repetitive sequences as compared to 11% of events integrated into rDNA and 70% into non-repetitive sequences in the wild-type cells. These results showed that deletion of RAD50 significantly changes the distribution of different classes of integration events, suggesting that Rad50 is required for nonhomologous integration at non-repetitive sequences more so than at repetitive ones. Furthermore, Southern analysis indicated that half of the events contained deletions at one or at both ends of the integrating DNA fragment, suggesting that Rad50 might have a role in protecting free ends of double-strand breaks. In contrast to the rad50∆ mutant, the rad50S mutant (separation of function allele) slightly increases the frequency of nonhomologous integration but the distribution of integration events is similar to that of wild-type cells with the majority of events integrated into a chromosomal locus. Our results suggest that deletion of RAD50 may block the major pathway of nonhomologous integration into a non-repetitive chromosomal locus and Rad50 may be involved in tethering two ends of the integrating DNA into close proximity that facilitates nonhomologous integration of both ends into a single chromosomal locus.     

The Drosophila Mre11/Rad50 complex is required to prevent both telomeric fusion and chromosome breakage

The MRN complex consists of the two evolutionarily conserved components Mre11 and Rad50 and the third less-conserved component Nbs1/Xrs2. This complex mediates telomere maintenance in addition to a variety of functions in response to DNA double-strand breaks, including homologous recombination, nonhomologous end joining (NHEJ), and activation of DNA damage checkpoints. Mutations in the Mre11 gene cause the human ataxia-telangiectasia-like disorder (ATDL). Here, we show that null mutations in the Drosophila mre11 and rad50 genes cause both telomeric fusion and chromosome breakage. Moreover, we demonstrate that these mutations are in the same epistasis group required for telomere capping and mitotic chromosome integrity. Using an antibody against Rad50, we show that this protein is uniformly distributed along mitotic chromosomes, and that Rad50 is unstable in the absence of its binding partner Mre11. To define the roles of rad50 and mre11 in telomere protection, mutant chromosome preparations were immunostained for both HP1 and HOAP, two proteins that protect Drosophila telomeres from fusion. Cytological analysis revealed that mutations in rad50 and mre11 drastically reduce accumulation of HOAP and HP1 at telomeres. This suggests that the MRN complex protects Drosophila telomeres by facilitating recruitment of HOAP and HP1 at chromosome ends.     

Structural and functional analysis of Mre11-3

The Mre11, Rad50 and Nbs1 proteins make up the conserved multi-functional Mre11 (MRN) complex involved in multiple, critical DNA metabolic processes including double-strand break repair and telomere maintenance. The Mre11 protein is a nuclease with broad substrate recognition, but MRN-dependent processes requiring the nuclease activity are not clearly defined. Here, we report the functional and structural characterization of a nuclease-deficient Mre11 protein termed mre11-3. Importantly, the hmre11-3 protein has wild-type ability to bind DNA, Rad50 and Nbs1; however, nuclease activity was completely abrogated. When expressed in cell lines from patients with ataxia telangiectasia-like disorder (ATLD), hmre11-3 restored the formation of ionizing radiation-induced foci. Consistent with the biochemical results, the 2.3 Å crystal structure of mre11-3 from Pyrococcus furiosus revealed an active site structure with a wild-type-like metal-binding environment. The structural analysis of the H85L mutation provides a detailed molecular basis for the ability of mre11-3 to bind but not hydrolyze DNA. Together, these results establish that the mre11-3 protein provides an excellent system for dissecting nuclease-dependent and independent functions of the Mre11 complex.     

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.     

The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability

The Mre11/Rad50/Nbs (MRN) complex and the two protein kinases ATM and ATR play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. It has been previously shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. Here, we have analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations.     

Most meiotic CAG repeat tract-length alterations in yeast are<Emphasis Type="Italic"> SPO11</Emphasis> dependent

The expansion of trinucleotide repeat sequences associated with hereditary neurological diseases is believed from earlier studies to be due to errors in DNA replication. However, more recent studies have indicated that recombination may play a significant role in triplet repeat expansion. CAG repeat tracts have been shown to induce double-strand breaks (DSBs) during meiosis in yeast, and DSB formation is dependent on the meiotic recombination machinery. The rate of meiotic instability is several fold higher than mitotic instability. To determine whether DSB repair is responsible for the high rate of repeat tract-length alterations, the frequencies of meiotic repeat-tract instability were compared in wild-type and spo11 mutant strains. In the spo11 background, the rate of meiotic repeat-tract instability remained at the mitotic level, suggesting that meiotic alterations of CAG repeat tracts in yeast occur by the recombination mechanism. Several of these meiotic tract-length alterations are due to DSB repair involving use of the sister chromatid as a template.     

Formation of the yeast Mre11-Rad50-Xrs2 complex is correlated with DNA repair and telomere maintenance

The yeast Mre11 is a multi-functional protein and is known to form a protein complex with Rad50 and Xrs2. In order to elucidate the relationship between Mre11 complex formation and its mitotic functions, and to determine domain(s) required for Mre11 protein interactions, we performed yeast two-hybrid and functional analyses with respect to Mre11 DNA repair and telomere maintenance. Evidence presented in this study indicates that the N-terminal region of Mre11 constitutes the core homo-dimerization and hetero-dimerization domain and is sufficient for Mre11 DNA repair and maintaining the wild-type telomere length. In contrast, a stretch of 134 amino acids from the extreme C-terminus, although essential for achieving a full level of self-association, is not required for the aforementioned Mre11 mitotic functions. Interestingly, deletion of these same 134 amino acids enhanced the interaction of Mre11 with Rad50 and Xrs2, which is consistent with the notion that this region is specific for meiotic functions. While Mre11 self-association alone is insufficient to provide the above mitotic activities, our results are consistent with a strong correlation between Mre11-Rad50-Xrs2 complex formation, mitotic DNA repair and telomere maintenance. This correlation was further strengthened by analyzing two mre11 phosphoesterase motif mutants ( mre11-2 and rad58S ), which are defective in DNA repair, telomere maintenance and protein interactions, and a rad50S mutant, which is normal in both complex formation and mitotic functions. Together, these results support and extend a current model regarding Mre11 structure and functions in mitosis and meiosis.     

Mre11 ATLD17/18 mutation retains Tel1/ATM activity but blocks DNA double-strand break repair

The Mre11 complex (Mre11-Rad50-Nbs1 or MRN) binds double-strand breaks where it interacts with CtIP/Ctp1/Sae2 and ATM/Tel1 to preserve genome stability through its functions in homology-directed repair, checkpoint signaling and telomere maintenance. Here, we combine biochemical, structural and in vivo functional studies to uncover key properties of Mre11-W243R, a mutation identified in two pediatric cancer patients with enhanced ataxia telangiectasia-like disorder. Purified human Mre11-W243R retains nuclease and DNA binding activities in vitro. X-ray crystallography of Pyrococcus furiosus Mre11 indicates that an analogous mutation leaves the overall Mre11 three-dimensional structure and nuclease sites intact but disorders surface loops expected to regulate DNA and Rad50 interactions. The equivalent W248R allele in fission yeast allows Mre11 to form an MRN complex that efficiently binds double-strand breaks, activates Tel1/ATM and maintains telomeres; yet, it causes hypersensitivity to ionizing radiation and collapsed replication forks, increased Rad52 foci, defective Chk1 signaling and meiotic failure. W248R differs from other ataxia telangiectasia-like disorder analog alleles by the reduced stability of its interaction with Rad50 in cell lysates. Collective results suggest a separation-of-function mutation that disturbs interactions amongst the MRN subunits and Ctp1 required for DNA end processing in vivo but maintains interactions sufficient for Tel1/ATM checkpoint and telomere maintenance functions.     

Genetic and biochemical evidences reveal novel insights into the mechanism underlying <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Sae2-mediated abrogation of DNA replication stress

In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in double-strand break (DSB) repair, replication stress and telomere length maintenance. Another protein linked to DSB repair is Sae2, which regulates MRX persistence at DSBs. However, very little is known about its role in DNA replication stress and repair. Here, we reveal a crucial role for Sae2 in DNA replication stress. We show that different mutant alleles of SAE2 cause hypersensitivity to genotoxic agents, and when combined with Δmre11 or nuclease-defective mre11 mutant alleles, the double mutants are considerably more sensitive suggesting that the sae2 mutations synergize with mre11 mutations. Biochemical studies demonstrate that Sae2 exists as a dimer in solution, associates preferentially with single-stranded and branched DNA structures, exhibits structure-specific endonuclease activity and cleaves these substrates from the 5′ end. Furthermore, we show that the nuclease activity is indeed intrinsic to Sae2. Interestingly, sae2^G270D protein possesses DNA-binding activity, but lacks detectable nuclease activity. Altogether, our data suggest a direct role for Sae2 nuclease activity in processing of the DNA structures that arise during replication and DNA damage and provide insights into the mechanism underlying Mre11-Sae2-mediated abrogation of replication stress-related defects in S. cerevisiae.     

Evidence for Distinct Functions of MRE11 in Arabidopsis Meiosis

The evolutionary conserved Mre11/Rad50/Nbs1 complex functions as one of the guardians of genome integrity in eukaryotes; it is required for the double-strand break repair, meiosis, DNA checkpoint, and telomere maintenance. To better understand the role of the MRE11 gene in Arabidopsis, we performed comparative analysis of several mre11 alleles with respect to genome stability and meiosis. The mre11-4 and mre11-2 alleles presumably produce truncated MRE11 proteins composed of the first 499 and 529 amino acids, respectively. Although the putative MRE11 truncated proteins differ only by 30 amino acids, the mutants exhibited strikingly different phenotypes in regards to growth morphology, genome stability and meiosis. While the mre11-2 mutants are fully fertile and undergo normal meiosis, the mre11-4 plants are sterile due to aberrant repair of meiotic DNA breaks. Structural homology analysis suggests that the T-DNA insertion in the mre11-4 allele probably disrupted the putative RAD50 interaction and/or homodimerization domain, which is assumed to be preserved in mre11-2 allele. Intriguingly, introgression of the atm-2 mutant plant into the mre11-2 background renders the double mutant infertile, a phenotype not observed in either parent line. This data indicate that MRE11 partially compensates for ATM deficiency in meiosis of Arabidopsis.     

Mre11-Rad50 Promotes Rapid Repair of DNA Damage in the Polyploid Archaeon Haloferax volcanii by Restraining Homologous Recombination

Polyploidy is frequent in nature and is a hallmark of cancer cells, but little is known about the strategy of DNA repair in polyploid organisms. We have studied DNA repair in the polyploid archaeon Haloferax volcanii, which contains up to 20 genome copies. We have focused on the role of Mre11 and Rad50 proteins, which are found in all domains of life and which form a complex that binds to and coordinates the repair of DNA double-strand breaks (DSBs). Surprisingly, mre11 rad50 mutants are more resistant to DNA damage than the wild-type. However, wild-type cells recover faster from DNA damage, and pulsed-field gel electrophoresis shows that DNA double-strand breaks are repaired more slowly in mre11 rad50 mutants. Using a plasmid repair assay, we show that wild-type and mre11 rad50 cells use different strategies of DSB repair. In the wild-type, Mre11-Rad50 appears to prevent the repair of DSBs by homologous recombination (HR), allowing microhomology-mediated end-joining to act as the primary repair pathway. However, genetic analysis of recombination-defective radA mutants suggests that DNA repair in wild-type cells ultimately requires HR, therefore Mre11-Rad50 merely delays this mode of repair. In polyploid organisms, DSB repair by HR is potentially hazardous, since each DNA end will have multiple partners. We show that in the polyploid archaeon H. volcanii the repair of DSBs by HR is restrained by Mre11-Rad50. The unrestrained use of HR in mre11 rad50 mutants enhances cell survival but leads to slow recovery from DNA damage, presumably due to difficulties in the resolution of DNA repair intermediates. Our results suggest that recombination might be similarly repressed in other polyploid organisms and at repetitive sequences in haploid and diploid species.