Spo11

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Scott Keeney - One of the best experts on this subject based on the ideXlab platform.

  • Structural and functional characterization of the Spo11 core complex
    Nature Structural & Molecular Biology, 2021
    Co-Authors: Corentin Claeys Bouuaert, Sam E. Tischfield, Eleni P. Mimitou, Ernesto Arias-palomo, James M. Berger, Scott Keeney
    Abstract:

    Biochemical and structural characterization of the meiotic DSB core complex of budding yeast reveals molecular architecture and DNA-binding properties similar to those of ancestral Topo VI. Spo11, which makes DNA double-strand breaks (DSBs) that are essential for meiotic recombination, has long been recalcitrant to biochemical study. We provide molecular analysis of Saccharomyces cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal topoisomerase VI, with Rec104 occupying a position similar to the Top6B GHKL-type ATPase domain. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), consistent with dimerization controlling DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex–duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles.

  • Structural and functional characterization of the Spo11 core complex
    2020
    Co-Authors: Corentin Claeys Bouuaert, Sam E. Tischfield, Eleni P. Mimitou, Ernesto Arias-palomo, James M. Berger, Scott Keeney
    Abstract:

    Spo11, which makes DNA double-strand breaks (DSBs) essential for meiotic recombination, is poorly understood mechanistically because it has been recalcitrant to biochemical study. Here, we provide a molecular analysis of S. cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal Topoisomerase VI, with Rec104 similar to a GHKL domain but without conserved ATPase motifs. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), indicating that dimerization may control DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex-duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing, and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles.

  • Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast
    Methods in molecular biology (Clifton N.J.), 2017
    Co-Authors: Isabel Lam, Neeman Mohibullah, Scott Keeney
    Abstract:

    Meiosis is a specialized form of cell division resulting in reproductive cells with a reduced, usually haploid, genome complement. A key step after premeiotic DNA replication is the occurrence of homologous recombination at multiple places throughout the genome, initiated with the formation of DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like protein Spo11. DSBs are distributed non-randomly in genomes, and understanding the mechanisms that shape this distribution is important for understanding how meiotic recombination influences heredity and genome evolution. Several methods exist for mapping where Spo11 acts. Of these, sequencing of Spo11-associated oligonucleotides (Spo11 oligos) is the most precise, specifying the locations of DNA breaks to the base pair. In this chapter we detail the steps involved in Spo11-oligo mapping in the SK1 strain of budding yeast Saccharomyces cerevisiae, from harvesting cells of highly synchronous meiotic cultures, through preparation of sequencing libraries, to the mapping pipeline used for processing the data.

  • Numerical and Spatial Patterning of Yeast Meiotic DNA Breaks by Tel1
    2016
    Co-Authors: Neeman Mohibullah, Scott Keeney
    Abstract:

    The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and distribution. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to examine the contribution of the DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM) to this regulation in Saccharomyces cerevisiae. A tel1Δ mutant had globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tel1 mutation also increased Spo11-oligonucleotide levels, but mutating known Tel1 phosphotargets on Hop1 and Rec114 did not. Deep sequencing of Spo11 oligonucleotides from tel1Δ mutants demonstrated that Tel1 shapes the nonrandom DSB distribution in ways that are distinct but partially overlapping with previously described contributions of the recombination regulator Zip3. Finally, we uncover a context-dependent role for Tel1 in hotspot competition, in which an artificial DSB hotspot inhibits nearby hotspots. Evidence for Tel1-dependent competition involving strong natural hotspots is also provided.

  • Mechanism and regulation of meiotic recombination initiation
    Cold Spring Harbor Perspectives in Biology, 2015
    Co-Authors: Isabel Lam, Scott Keeney
    Abstract:

    Meiotic recombination involves the formation and repair of programmed DNA double-strand breaks (DSBs) catalyzed by the conserved Spo11 protein. This review summarizes recent studies pertaining to the formation of meiotic DSBs, including the mechanism of DNA cleavage by Spo11, proteins required for break formation, and mechanisms that control the location, timing, and number of DSBs. Where appropriate, findings in different organisms are discussed to highlight evolutionary conservation or divergence.

Yukiko Tokuyama - One of the best experts on this subject based on the ideXlab platform.

  • Class switch recombination signals induce lymphocyte-derived Spo11 expression and Spo11 antisense oligonucleotide inhibits class switching.
    Cellular immunology, 2001
    Co-Authors: Haruhiko Tokuyama, Yukiko Tokuyama
    Abstract:

    Recently, we showed that mouse Spo11 is induced in normal mu(+) B cells by class switch recombination (CSR) stimuli, by RT-PCR using primers based on the reported cDNA sequence of testis-derived Spo11 (test-Spo11) cDNA. In the present study, we first determined the cDNA sequence of lymphocyte-derived Spo11 (lym-Spo11). The 5' upstream portion had an as yet unreported sequence but the remaining part from exons 2 to 12 and the subsequent 3'UTR was completely identical to that of test-Spo11. RT-PCR analysis indicated that lymphocytes express lym-Spo11 but not test-Spo11. Second, we showed that lym-Spo11 is strongly induced (above eightfold) in the IgA CSR system of LPS-stimulated mu(+)B cells in the presence of all-trans retinoic acid and IL-4. Finally, we examined whether lym-Spo11 antisense S-oligonucleotide (AS) can inhibit CSR reactions in three in vitro CSR systems, IgA,IgG1, and IgE. Lym-Spo11 AS or the sense oligonucleotide was added to the cultures at the start, and total RNA was extracted after 4 days. IgA, IgG1, and IgE mRNAs (J(H)C(H)) and mature germline C(H) transcripts (I(H)C(H)) were quantitatively assayed by RT-PCR. AS inhibited J(H)C(H) expression dose-dependently. In all three systems, the maximum inhibition by 20 microM AS was in the range of 60 to 90%. Interestingly, I(H)C(H) was also inhibited by AS to a similar extent as J(H)C(H). These results suggested that lym-Spo11 plays an important role in the initiation step of CSR.

  • Mouse Homolog of Saccharomyces cerevisiae Spo11 Is Induced in Normal μ(+)B-Cells by Stimuli That Cause Germline CH Transcription and Subsequent Class Switch Recombination
    Cellular immunology, 2000
    Co-Authors: Haruhiko Tokuyama, Yukiko Tokuyama
    Abstract:

    Abstract The first step of Ig heavy chain class switch recombination (CSR) is considered to be DNA double strand break (DSB) formation in the two switch (S) regions (S μ and downstream S H ), although the underlying mechanism is unknown. Recently, it has been demonstrated that at least Spo11, a homolog of the novel type II topoisomerase (topo VI) that catalyzes DSB formation, is involved in the initiation of meiotic recombination of Saccaromyces cerevisiae. In the present study, we examined whether the mouse homolog of Spo11 is induced in normal mouse μ(+)B-cells by stimuli that cause an early step of CSR, germline C H transcription, and subsequent CSR. Two CSR systems were used: IgA CSR induced by all-trans retinoic acid, IL-5, and LPS, and IgG1 CSR induced by IL-4 and LPS. Germline transcript and mouse Spo11 expression were analyzed by RT-PCR. In both systems, first germline transcripts were clearly detected on day 2 and then Spo11 was detected on day 3, increasing thereafter with time. The time course of changes in Spo11 expression coincided with that of CSR. Spo11 seems to be induced by CSR-inducing stimuli, regardless of the direction of CSR. These results suggested that mouse Spo11 might participate in the initiation step of CSR.

  • mouse homolog of saccharomyces cerevisiae Spo11 is induced in normal μ b cells by stimuli that cause germline ch transcription and subsequent class switch recombination
    Cellular Immunology, 2000
    Co-Authors: Haruhiko Tokuyama, Yukiko Tokuyama
    Abstract:

    Abstract The first step of Ig heavy chain class switch recombination (CSR) is considered to be DNA double strand break (DSB) formation in the two switch (S) regions (S μ and downstream S H ), although the underlying mechanism is unknown. Recently, it has been demonstrated that at least Spo11, a homolog of the novel type II topoisomerase (topo VI) that catalyzes DSB formation, is involved in the initiation of meiotic recombination of Saccaromyces cerevisiae. In the present study, we examined whether the mouse homolog of Spo11 is induced in normal mouse μ(+)B-cells by stimuli that cause an early step of CSR, germline C H transcription, and subsequent CSR. Two CSR systems were used: IgA CSR induced by all-trans retinoic acid, IL-5, and LPS, and IgG1 CSR induced by IL-4 and LPS. Germline transcript and mouse Spo11 expression were analyzed by RT-PCR. In both systems, first germline transcripts were clearly detected on day 2 and then Spo11 was detected on day 3, increasing thereafter with time. The time course of changes in Spo11 expression coincided with that of CSR. Spo11 seems to be induced by CSR-inducing stimuli, regardless of the direction of CSR. These results suggested that mouse Spo11 might participate in the initiation step of CSR.

Haruhiko Tokuyama - One of the best experts on this subject based on the ideXlab platform.

  • Class switch recombination signals induce lymphocyte-derived Spo11 expression and Spo11 antisense oligonucleotide inhibits class switching.
    Cellular immunology, 2001
    Co-Authors: Haruhiko Tokuyama, Yukiko Tokuyama
    Abstract:

    Recently, we showed that mouse Spo11 is induced in normal mu(+) B cells by class switch recombination (CSR) stimuli, by RT-PCR using primers based on the reported cDNA sequence of testis-derived Spo11 (test-Spo11) cDNA. In the present study, we first determined the cDNA sequence of lymphocyte-derived Spo11 (lym-Spo11). The 5' upstream portion had an as yet unreported sequence but the remaining part from exons 2 to 12 and the subsequent 3'UTR was completely identical to that of test-Spo11. RT-PCR analysis indicated that lymphocytes express lym-Spo11 but not test-Spo11. Second, we showed that lym-Spo11 is strongly induced (above eightfold) in the IgA CSR system of LPS-stimulated mu(+)B cells in the presence of all-trans retinoic acid and IL-4. Finally, we examined whether lym-Spo11 antisense S-oligonucleotide (AS) can inhibit CSR reactions in three in vitro CSR systems, IgA,IgG1, and IgE. Lym-Spo11 AS or the sense oligonucleotide was added to the cultures at the start, and total RNA was extracted after 4 days. IgA, IgG1, and IgE mRNAs (J(H)C(H)) and mature germline C(H) transcripts (I(H)C(H)) were quantitatively assayed by RT-PCR. AS inhibited J(H)C(H) expression dose-dependently. In all three systems, the maximum inhibition by 20 microM AS was in the range of 60 to 90%. Interestingly, I(H)C(H) was also inhibited by AS to a similar extent as J(H)C(H). These results suggested that lym-Spo11 plays an important role in the initiation step of CSR.

  • Mouse Homolog of Saccharomyces cerevisiae Spo11 Is Induced in Normal μ(+)B-Cells by Stimuli That Cause Germline CH Transcription and Subsequent Class Switch Recombination
    Cellular immunology, 2000
    Co-Authors: Haruhiko Tokuyama, Yukiko Tokuyama
    Abstract:

    Abstract The first step of Ig heavy chain class switch recombination (CSR) is considered to be DNA double strand break (DSB) formation in the two switch (S) regions (S μ and downstream S H ), although the underlying mechanism is unknown. Recently, it has been demonstrated that at least Spo11, a homolog of the novel type II topoisomerase (topo VI) that catalyzes DSB formation, is involved in the initiation of meiotic recombination of Saccaromyces cerevisiae. In the present study, we examined whether the mouse homolog of Spo11 is induced in normal mouse μ(+)B-cells by stimuli that cause an early step of CSR, germline C H transcription, and subsequent CSR. Two CSR systems were used: IgA CSR induced by all-trans retinoic acid, IL-5, and LPS, and IgG1 CSR induced by IL-4 and LPS. Germline transcript and mouse Spo11 expression were analyzed by RT-PCR. In both systems, first germline transcripts were clearly detected on day 2 and then Spo11 was detected on day 3, increasing thereafter with time. The time course of changes in Spo11 expression coincided with that of CSR. Spo11 seems to be induced by CSR-inducing stimuli, regardless of the direction of CSR. These results suggested that mouse Spo11 might participate in the initiation step of CSR.

  • mouse homolog of saccharomyces cerevisiae Spo11 is induced in normal μ b cells by stimuli that cause germline ch transcription and subsequent class switch recombination
    Cellular Immunology, 2000
    Co-Authors: Haruhiko Tokuyama, Yukiko Tokuyama
    Abstract:

    Abstract The first step of Ig heavy chain class switch recombination (CSR) is considered to be DNA double strand break (DSB) formation in the two switch (S) regions (S μ and downstream S H ), although the underlying mechanism is unknown. Recently, it has been demonstrated that at least Spo11, a homolog of the novel type II topoisomerase (topo VI) that catalyzes DSB formation, is involved in the initiation of meiotic recombination of Saccaromyces cerevisiae. In the present study, we examined whether the mouse homolog of Spo11 is induced in normal mouse μ(+)B-cells by stimuli that cause an early step of CSR, germline C H transcription, and subsequent CSR. Two CSR systems were used: IgA CSR induced by all-trans retinoic acid, IL-5, and LPS, and IgG1 CSR induced by IL-4 and LPS. Germline transcript and mouse Spo11 expression were analyzed by RT-PCR. In both systems, first germline transcripts were clearly detected on day 2 and then Spo11 was detected on day 3, increasing thereafter with time. The time course of changes in Spo11 expression coincided with that of CSR. Spo11 seems to be induced by CSR-inducing stimuli, regardless of the direction of CSR. These results suggested that mouse Spo11 might participate in the initiation step of CSR.

Maria Jasin - One of the best experts on this subject based on the ideXlab platform.

  • ATM controls meiotic double-strand-break formation
    Nature, 2011
    Co-Authors: Julian Lange, Maria Jasin, Jing Pan, Michael P. Thelen, Francesca Cole, Scott Keeney
    Abstract:

    In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the Spo11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation. However, improperly repaired DSBs can cause meiotic arrest or mutation; thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of Spo11 protein ever makes a DSB in yeast or mouse and Spo11 and its accessory factors remain abundant long after most DSB formation ceases, implying the existence of mechanisms that restrain Spo11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signalling and promote DSB repair. Levels of Spo11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold in spermatocytes lacking ATM. Moreover, Atm mutation renders Spo11-oligonucleotide levels sensitive to genetic manipulations that modulate Spo11 protein levels. We propose that ATM restrains Spo11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency.

  • Mice deficient for the type II topoisomerase-like DNA transesterase Spo11 show normal immunoglobulin somatic hypermutation and class switching.
    European journal of immunology, 2002
    Co-Authors: Ulf Klein, Scott Keeney, Frédéric Baudat, Gloria Esposito, Maria Jasin
    Abstract:

    Somatic hypermutation in B cells undergoing T cell dependent immune responses generates high affinity antibodies that provide protective immunity. B cells also switch from the expression of immunoglobulin (Ig) M and IgD to that of other Ig classes through somatic DNA recombination. Recent work has implicated DNA strand breaks, possibly DNA double strand breaks (DSB), as the initiating lesions in both class switch recombination and hypermutation, although the etiology of these lesions is not understood. Spo11, a protein structurally related to archaeal type II topoisomerases, generates DSB that initiate meiotic recombination. This characteristic, together with its expression pattern, marks this enzyme as a potential candidate for the initiation of hypermutation, and perhaps also for Ig class switching. To investigate whether Spo11 is involved in these processes, we studied the T cell dependent immune response of Spo11-deficient (Spo11(-/-)) mice against the hapten nitrophenyl (NP). We found that V186.2-bearing IgG1 transcripts had normal levels and patterns of somatic hypermutation. Furthermore, Spo11(-/-) mice showed normal serum levels of all Ig isotypes. These results indicate that Spo11 is not required for Ig hypermutation or class switch recombination.

  • Recombinational DNA double-strand breaks in mice precede synapsis
    Nature Genetics, 2001
    Co-Authors: Shantha K Mahadevaiah, Josefa Blanco-rodríguez, Scott Keeney, Frédéric Baudat, James M A Turner, Emmy P Rogakou, Peter De Boer, Maria Jasin, William M. Bonner, Paul S. Burgoyne
    Abstract:

    In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand breaks (DSBs), a process that precedes homologous synapsis. Here we use an antibody specific for a phosphorylated histone (gamma-H2AX, which marks the sites of DSBs) to investigate the timing, distribution and Spo11-dependence of meiotic DSBs in the mouse. We show that, as in yeast, recombination in the mouse is initiated by Spo11-dependent DSBs that form during leptotene. Loss of gamma-H2AX staining (which in irradiated somatic cells is temporally linked with DSB repair) is temporally and spatially correlated with synapsis, even when this synapsis is 'non-homologous'.

  • Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11
    Molecular Cell, 2000
    Co-Authors: Frédéric Baudat, Julie Pui Yuen, Maria Jasin, Katia Manova, Scott Keeney
    Abstract:

    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.

Frank Hartung - One of the best experts on this subject based on the ideXlab platform.

  • analysis of Spo11 protein interaction during meiosis
    Achtes Nachwuchswissenschaftlerforum 2015 : 19.-21. Oktober in Quedlinburg ; Abstracts, 2015
    Co-Authors: Thorben Sprink, Frank Hartung, Joachim Schiemann
    Abstract:

    Meiosis as the specialized cell division of sexual reproduction plays a crucial role in the exchange and reorganization of genetic material between two individuals by dividing the chromosome set in half and forming gametes. Even though in the last years major findings in the field of meiosis have been achieved, especially in plants, some key questions remain concealed. For a proper meiosis the initiation of double strand breaks (DSBs) during early prophase I is essential. Without DSBs no physical connection can occur between homologous chromosomes and recombination, pairing, and crossing over are excluded. So far in all analyzed eukaryotes Spo11, a meiosis specific transesterase, is the key enzyme inducing DSBs. But other than in animals and fungi where a single Spo11 is sufficient, plants need at least two different Spo11, referred to as Spo11-1 and Spo11-2, for proper meiosis. In Arabidopsis thaliana both have crucial functions and are essential in a functional form for the induction of meiotic DSBs as single knock out mutants are leading to near sterility by random chromosome distribution. Despite the same function of the homologs Spo111 and -2, the identity between both proteins is quite low. Homology of the orthologous Spo11 from different organisms is much higher. By exchanging Spo11-1 and -2 in Arabidopsis by their orthologs from various organisms we could demonstrate a species specific function of each Spo11, as a functional complementation of sterility could only be achieved with Spo11 from closely related species from the Brassicaceae. By exchanging non conserved regions between Spo11-1 and -2 of Arabidopsis we additionally could show a sequence specific function for each Spo11, as a functional rescue could not be achieved with all chosen regions. Interestingly, we could reveal a specific pattern of aberrant spliced isoforms for each Spo11 which are also sequence as well as species specific. By producing antibodies against AthSpo11-1 and -2 we were able to analyze for the first time the binding of Spo11-2 onto the DNA and perform co-immunolocalization studies with Spo11-1 and -2.

  • The splicing fate of plant Spo11 genes.
    Frontiers in plant science, 2014
    Co-Authors: Thorben Sprink, Frank Hartung
    Abstract:

    Towards the global understanding of plant meiosis, it seems to be essential to decipher why all as yet sequenced plants need or at least encode for two different meiotic Spo11 genes. This is in contrast to mammals and fungi, where only one Spo11 is present. Both Spo11 in plants are essential for the initiation of double strand breaks (DSBs) during the meiotic prophase. In nearly all eukaryotic organisms DSB induction by Spo11 leads to meiotic DSB repair, thereby ensuring the formation of a necessary number of crossovers (CO) as physical connections between the allelic chromosomes. We aim to investigate the specific functions and evolution of both Spo11 genes in land plants. Therefore, we identified and cloned the respective orthologous genes from Brassica rapa, Carica papaya, Oryza sativa and Physcomitrella patens. In parallel we determined the full length cDNA sequences of Spo11-1 and -2 from all of these plants by RT-PCR. During these experiments we observed that the analyzed plants exhibit a pattern of aberrant splicing products of both Spo11 mRNAs. Such an aberrant splicing has previously been described for Arabidopsis and therefore seems to be conserved throughout evolution. Most of the splicing forms of Spo11-1 and -2 seem to be non functional as they either showed intron retention or shortened exons accompanied by a frameshift leading to premature termination codons (PTCs) in most cases. Nevertheless, we could detect one putative functional alternatively spliced mRNA for Spo11-1 and -2 each, indicating that splicing of Spo11 does not depend only on the gene sequence but also on the plant species and that it might play a regulatory role.

  • Initiation of meiotic double strand breaks in plants depends on two different Spo11 proteins
    2011
    Co-Authors: Thorben Sprink, Frank Hartung
    Abstract:

    The pairing and balanced distribution of allelic chromosomes is one of the main factors leading to genetic diversity and a successful meiotic outcome in eukaryotes. This process depends in nearly all analyzed eukaryotic organisms on the initiation of double strand breaks (DSBs) by the protein Spo11, an evolutionary conserved meiotic transesterase. Spo11 is introducing these DSBs during early meiotic prophase. Whereas in animals and fungi only a single Spo11 is present, plants have at least two Spo11 proteins which are active and essential in meiosis (Spo11-1 and Spo11-2). Single knockout mutants of Arabidopsis Spo11-1 as well as Spo11-2 are nearly sterile and random chromosome segregation during meiosis occurs. In all so far sequenced plants genes orthologous to Arabidopsis Spo11-1 and -2 exist. Our aims are to investigate whether the function of these two different Spo11 proteins is species specific or interchangeable between near and far distantly related plants. Furthermore, we want to define which regions of the proteins determine the specificity of the respective Spo11 protein 1 or 2. To analyze the functional conservation we used orthologous genes from different land plants (Brassica rapa , Carica papaya , Oryza sativa and   Physcomitrella patens ) for heterologous complementation of well characterized Spo11 mutants from Arabidopsis. To figure out which part of the protein sequence determines the specificity, we interchanged regions between the two Spo11 genes in Arabidopsis and transformed mutant plants with these swap constructs. Here we will present first results on heterologous complementation with near related land plants as well as first results of interchanged regions between the two Spo11 proteins. We will show chromosome distribution as well as pollen vitality by different microscopic techniques. We will also evaluate if there is a differences in the number of seeds produced by the different complemented plants. Analysing the results of these experiments, we should be able to answer the questions if there are sequence and/or species specific functions of each Spo11 and which region(s) of the proteins are essential for the initiation of meiotic double strand breaks.

  • The Catalytically Active Tyrosine Residues of Both Spo11-1 and Spo11-2 Are Required for Meiotic Double-Strand Break Induction in Arabidopsis
    The Plant Cell, 2007
    Co-Authors: Frank Hartung, Rebecca Wurz-wildersinn, Stefanie Suer, Ingo Schubert, Jörg Fuchs, Holger Puchta
    Abstract:

    Spo11, a homolog of the subunit A of the archaebacterial topoisomerase VI, is essential for double-strand break (DSB)– induced initiation of meiotic recombination. In contrast with single homologs in animals and yeasts, three homologs are present in Arabidopsis thaliana and other higher plants. Whereas At Spo11-3 is involved in somatic endoreduplication, At Spo11-1 and, as recently shown, At Spo11-2 are essential for the initiation of meiotic recombination. Further defining the role of At Spo11-2, we were able to demonstrate that it is required for proper chromosome segregation, as its loss resulted in aneuploidy in the surviving progeny. The double mutant Spo11-1 Spo11-2 does not differ phenotypically from the single mutants, indicating that both proteins are required for the same step. Contrary to the observations for the At rad51-1 single mutant, the combination of Spo11-2 and rad51-1 did not lead to chromosome fragmentation, indicating that Spo11-2, like Spo11-1, is required for DSB induction. As the meiotic phenotype of both single Spo11 mutants can be reversed by complementation using the full-length genes but not the same constructs mutated in their respective catalytically active Tyr, both proteins seem to participate directly in the DNA breakage reaction. The active involvement of two Spo11 homologs for DSB formation reveals a striking difference between plants and other eukaryotes in meiosis.

  • Molecular characterization of homologues of both subunits A (Spo11) and B of the archaebacterial topoisomerase 6 in plants.
    Gene, 2001
    Co-Authors: Frank Hartung, Holger Puchta
    Abstract:

    Abstract The Spo11 protein is an eukaryotic homologue of the topoisomerase 6 subunit A from archaebacteria. In yeast Spo11p has been found to bind covalently to double-strand breaks (DSBs) during meiosis. Single homologues of the Spo11 gene exist in various eukaryotes, except plants. Previously, we found in the Arabidopsis thaliana genome two ancient paralogs, AtSpo11-1 and 2. Here we report on the molecular characterization of a third one, AtSpo11-3. This puzzling finding might be explained by the fact that we detected additionally – for the first time outside of the archaebacterial kingdom – a homologue of the subunit B of topoisomerase 6, AtTOP6B. Both AtSpo11-3 and AtTOP6B are abundantly expressed in Arabidopsis and EST comparisons indicate the presence of both genes in various plant species. Via two hybrid studies we could demonstrate that full length AtTop6B is able to interact with AtSpo11-2 and 3 but not with AtSpo11-1. Our data suggest that plants possess in contrast to other eukaryotes an additional archaebacterial kind of topoisomerase.