Research Article

The TopoVIB-Like protein family is required for meiotic DNA double-strand break formation

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Science  26 Feb 2016:
Vol. 351, Issue 6276, pp. 943-949
DOI: 10.1126/science.aad5309

A partner protein for meiotic snip

Eukaryotes generate germ cells through meiotic recombination. This process initiates through breaks in genomic DNA catalyzed by the SPO11 protein. Vrielynck et al. and Robert et al. discover that SPO11, like topoisomerase VI enzymes, interacts with a partner protein (see the Perspective by Bouuaert and Keeney). This partner is required for proper meiotic recombination and is found in a wide range of eukaryotes, suggesting that it is a universal feature of the essential recombination step.

Science, this issue p. 939, 943; see also p. 916

Abstract

Meiotic recombination is induced by the formation of DNA double-strand breaks (DSBs) catalyzed by SPO11, the ortholog of subunit A of TopoVI DNA topoisomerase (TopoVIA). TopoVI activity requires the interaction between A and B subunits. We identified a conserved family of plant and animal proteins [the TOPOVIB-Like (TOPOVIBL) family] that share strong structural similarity to the TopoVIB subunit of TopoVI DNA topoisomerase. We further characterize the meiotic recombination proteins Rec102 (Saccharomyces cerevisiae), Rec6 (Schizosaccharomyces pombe), and MEI-P22 (Drosophila melanogaster) as homologs to the transducer domain of TopoVIB. We demonstrate that the mouse TOPOVIBL protein interacts and forms a complex with SPO11 and is required for meiotic DSB formation. We conclude that meiotic DSBs are catalyzed by a complex involving SPO11 and TOPOVIBL.

Sexual reproduction involves the formation of haploid gametes through a specialized cell cycle called meiosis. In most species, proper chromosome segregation at the first meiotic division requires connections between homologous chromosomes mediated by meiotic recombination. Meiotic recombination is initiated by programmed double-strand break (DSB) induction at the beginning of meiotic prophase and involves several evolutionarily conserved genes (1), including Spo11. This gene encodes a protein predicted to catalyze DSB formation, on the basis of two observations: (i) the similarity between SPO11 and the catalytic subunit of the TopoVI family of type II DNA topoisomerases (TopoVIA), including the presence of an essential and conserved tyrosine involved in the transesterification reaction for DNA break formation (2), and (ii) the detection of covalent SPO11-DNA intermediates in meiotic cells (3, 4). TopoVI belongs to the type IIB family of topoisomerases, and its activity promotes relaxation of negative and positive supercoiled DNA and DNA decatenation through cleavage and ligation cycles (5, 6). TopoVI is composed of two subunits (A and B) that form a heterotetramer. Subunit B (TopoVIB) contains an adenosine triphosphate (ATP) binding domain and a transducer domain that communicates with the ATP binding site and interacts with TopoVIA (7, 8). DNA cleavage by TopoVI requires both subunits and ATP binding (9). Subunit B, through ATP binding and hydrolysis, is involved in the conformational change of the complex and regulates the interaction between TopoVIA and TopoVIB homodimers for strand passage. The specific properties of this complex mechanism that are conserved in SPO11-catalyzed DSB formation remain unknown. Moreover, whether SPO11 functions alone or through association with another subunit has remained an open question for the past 18 years.

A conserved family of TopoVIB-Like proteins

While searching for a mammalian ortholog of the MTOPVIB gene, which is required for meiotic DSB formation in Arabidopsis thaliana (10), we identified a family of proteins sharing similarity with TopoVIB from archaea to chordates. Specifically, we performed PSI-BLAST (Position-Specific Iterative Basic Local Alignment Search Tool) searches, using A. thaliana MTOPVIB (AtMTOPVIB) as a query, to identify the product of the mouse gene Gm960 (homolog of human C11orf80) that has four small regions displaying strong similarity to AtMTOPVIB (fig. S1A). Further alignments along full-length plant and mammalian proteins (fig. S1B) and analysis using HHpred (http://toolkit.tuebingen.mpg.de/hhpred) for detecting structural conservation enabled us to identify archaeal TopoVIB members with high significance (P = 99.1%, Expect value E = 3.4 × 10–9 for Sulfolobus shibatae). We named this protein family TopoVIB-Like (TopoVIBL), with plant and animal (chordates, ascidians, mollusks, and sea urchins) members (fig. S2) qualified on the basis of the conservation of the main TopoVIB domains: the GHKL domain (Bergerat fold), found in the GHKL (Gyrase, Hsp90, Histidine kinase, MutL) superfamily of adenosine triphosphatases (ATPases) (11), and the transducer domain (7, 8) (Figs. 1 and 2 and fig. S1C). Despite an overall low level of identity and similarity [11 and 19%, respectively, between Mus musculus TOPOVIBL (encoded by Gm960, hereafter named Top6bl) and S. shibatae TopoVIB over 140 residues], key motifs (N box and the G1, G2, and G3 glycine-containing motifs) were identified within the GHKL domain, and the predicted secondary structure was conserved (Fig. 1A and fig. S3A). The Bergerat fold motifs play an important role in coordinating interaction with Mg2+ ions and with ATP; additionally, these motifs provide flexibility to the structure for ATP binding. On the basis of protein alignments, we predicted the structure of mouse TOPOVIBL that shares strong similarity with that of S. shibatae TopoVIB (Fig. 1B and fig. S3B) [root mean square deviation (RMSD) = 1.75 Å over 140 Cα atoms]. Comparison of the two structures highlighted the conservation of the ATP lid (a flexible loop that coordinates ATP binding), including a poorly conserved G2 motif. Some TopoVIB structural elements—such as the N-terminal helix (involved in dimerization) and helix α3 (12)—were not detected in the TopoVIBL family, suggesting potential differences in the activity of the GHKL domain.

Fig. 1 TopoVIBL contains the conserved motifs and structure of the GHKL domain.

(A) GHKL domain conservation among the B subunits of type IIB topoisomerases. Images show alignment of the sequences of the archaeal S. shibatae (Sulf) and Methanosarcina mazei (Meth) TopoVIB ATPase domains with their plant and mammalian TopoVIBL counterparts. AtMTOPVIB: A. thaliana; Os: Oryza sativa; Hs: Homo sapiens C11orf80; MmTOPOVIBL: M. musculus Gm960 product. The secondary structural elements of SulfTopoVIB (Protein Data Bank ID: 2zbk_B) are shown above the alignment. The four core elements of the ATP-binding Bergerat fold (11) are indicated by red dashed boxes. Fully conserved amino acid residues are depicted in white on a red background, whereas partially conserved residues are in bold within yellow boxes (18). (B) Comparison of the models of the mouse TOPOVIBL GHKL and transducer domain structures with the S. shibatae TopoVIB structure from which they were derived. Orange, β strands; blue, α helices; pink, ATP lid; green, switch loops; red, helical regions predicted to interact with mouse SPO11.

Fig. 2 The transducer domain of TopoVIBL and its interaction with Spo11 are widely conserved.

(A) Transducer domain conservation. Images show alignment of the sequences of the long α-helical regions of archaeal TopoVIB transducer domains with their plant, fungal, and metazoan TopoVIBL counterparts. The secondary structural elements of SulfTopoVIB are shown above the alignment. The positions of the conserved WOC motif and the switch loop are indicated. Black ovals indicate the S. shibatae residues involved in the interaction with SulfTopoVIA. Zm, Zea mays; Ri, R. irregularis; Sp, S. pombe; Sc, S. cerevisiae; Dm, D. melanogaster; Spu, Strongylocentrotus purpuratus; Dr, Danio rerio. Other abbreviations and symbols are as in Fig. 1. (B) Comparison of the archaeal TopoVIA-B heterodimer structure (left) to mouse TOPOVIBL-SPO11β (middle) and yeast Rec102-Spo11 (right) models. Green, GHKL domain; red, transducer domain; blue, TopoVIA/Spo11.

The transducer domain in the C-terminal region of TopoVIBL proteins also displayed marked similarity (conserved motifs or residues and structural elements) to that of TopoVIB, despite an overall high level of sequence divergence (11% identity and 22% similarity between mouse TOPOVIBL and S. shibatae TopoVIB over 206 residues) (Fig. 2A and fig. S4A). In particular, we detected stronger conservation at a motif that we named WOC (for WKxY-containing octapeptide) and over the C-terminal helices α11 and α12 that, in TopoVIB, interact with TopoVIA (7, 8) (Fig. 2, A and B). A flexible loop was also predicted on the modeled structure of mouse TOPOVIBL in the region corresponding to the switch loop of TopoVIB that interacts with ATP bound to the GHKL domain, thus regulating phosphate release and complex conformation (12) (fig. S4B) (RMSD = 1.78 Å over 206 Cα atoms). The overall organization of the GHKL and transducer domains (the two primary domains of mouse TOPOVIBL) indicates that this protein shares structural similarity with S. shibatae TopoVIB, except for the intermediate helix–two turns–helix (H2TH) domain with unknown function (fig. S5, A and B). We also detected strong divergence at the C terminus of the proteins (C-terminal domain; see fig. S1B) after the two conserved helices of the transducer domain, with extensions of variable length among species.

Further PSI-BLAST searches for homologs in fungi led us to the identification of a predicted protein from the glomerale Rhizophagus irregularis, with conserved motifs overlapping the WOC motif and counterparts of archaeal β strand 14, as well as helices α11 and α12 described above, but without an obvious GHKL domain (fig. S6, A and B). The glomerale protein was then incorporated in a series of PSI-BLAST searches that identified fungal homologs in mucorales, basidiomycetes, and ascomycetes [including Saccharomyces cerevisiae (Rec102) and Schizosaccharomyces pombe (Rec6)], which contained the WOC motif, the switch loop, and the α11 and α12 helices (Fig. 2A and figs. S7, A and C, and S8). Rec102 and Rec6 (new annotation in fig. S7B) are required for meiotic DSB formation (1) and physically interact with Spo11 (1315). This interaction is supported by the predicted structure of the Rec102-Spo11 complex (Fig. 2B). Additional PSI-BLAST searches and secondary structure predictions enabled detection of significant structural homologies between the TopoVIBL transducer domain and the evolutionarily conserved C-terminal region of Drosophila melanogaster MEI-P22 (fig. S9), which is also required for meiotic DSB formation (16). The absence of a detectable GHKL-type ATPase domain in fungal and insect homologs indicates that an alternative protein substitutes for this domain or that the molecular mechanism of DSB formation displays distinct properties in these species. Overall, structural modeling predicts an interaction between TopoVIBL and SPO11 to form a heterodimer (Fig. 2B), and potentially a heterotetramer, for DSB formation.

Mouse TOPOVIBL and SPO11 interact

The conserved features and the structure analyses presented above predict that TOPOVIBL interacts directly with SPO11. In mice, there are two major SPO11 splice variants (α and β). SPO11β contains an additional 38 amino acids in the N terminus that correspond to the TopoVIA region interacting with TopoVIB (7, 8). Analysis of mice expressing only Spo11β showed that this isoform is proficient in meiotic DSB formation (17). Using the yeast two-hybrid assay, we found that mouse TOPOVIBL interacts with SPO11β but not with SPO11α. This highlights the essential role of the aforementioned SPO11β 38–amino acid region, which overlaps with the N-terminal α helix, in such interaction. By deletion analysis, we also determined that the TOPOVIBL C-terminal region, which includes the transducer domain, is involved in the interaction (Fig. 3A and figs. S10 and S11). Moreover, coexpression of TOPOVIBL and SPO11β in Escherichia coli suggested the formation of a complex. Affinity purification of His-tagged TOPOVIBL allowed the copurification of TOPOVIBL with SPO11β but not with SPO11α and was dependent on the presence of the tag used (Fig. 3B, top panels). Furthermore, upon gel filtration chromatography and glycerol gradient sedimentation, the size of the complex including TOPOVIBL and SPO11β was estimated at 150 to 250 kDa, compatible with the assembly of a heterotetramer (217 kDa) (Fig. 3B, bottom panel, and fig. S12).

Fig. 3 Mouse TOPOVIBL interacts with SPO11 and is expressed in testis upon entry into meiosis.

(A) TOPOVIBL C-terminal region interacts with SPO11β in a yeast two-hybrid assay. TOPOVIBL and SPO11 were fused to Gal4AD (AD) and Gal4BD (BD), respectively. The 38 residues specific to SPO11β are shown in orange, and the positions of the GHKL and transducer domains are indicated above full-length TOPOVIBL. – denotes no interaction; + indicates interaction. Aa, amino acids. (B) TOPOVIBL and SPO11β form a soluble complex of 150 to 250 kDa. (Top panels) His6-TOPOVIBL and SPO11β or SPO11α, as well as His6-Flag-TOPOVIBL and SPO11β, were coexpressed in E. coli and affinity-purified. P, pellet; Sn, supernatant; E, nickel nitrilotriacetic acid (NiNTA) elution; (E), concentrated NiNTA elution; FT, flow-through; E′, Flag elution. (Bottom panel) His6-Flag-TOPOVIBL and SPO11β were affinity-purified, and proteins were analyzed by gel filtration chromatography. Anti-His or anti-SPO11 antibodies were used for detections. (C) Top6bl expression by RT-PCR using RNA extracted from various adult mouse tissues. Control, sample without RNA; RT with (+) or without (–) reverse transcriptase. Actin was used as a positive control of PCR amplification (bottom panel). (D) RT-PCR analysis of Top6bl (top panel) and Spo11β and Spo11α (middle panel) in RNA from testes of mice at various ages (4 to 30 dpp). (Bottom panel) Controls were as in (C).

Top6bl is required for meiotic DSB formation

To evaluate Top6bl biological function during meiosis, we first assessed its expression in tissues from adult mice. Top6bl was expressed in testis but not in other tissues (Fig. 3C and fig. S13A). The temporal expression profile of genes during meiosis can be determined by taking advantage of the synchronous first wave of entry of spermatogonia into meiosis, which takes place ~8 days postpartum (dpp). Spermatocytes then progress into meiotic prophase from 10 to 20 dpp and reach the first meiotic division around 21 dpp. Top6bl expression could not be detected in testis RNA from mice on 4 and 6 dpp but only from 8 dpp onward, with a peak of expression around 12 to 14 dpp, at the beginning of meiotic prophase. This temporal pattern is similar to that of Spo11β expression (Fig. 3D). In female mice, Top6bl transcripts were detected in embryonic ovaries when oocytes proceeded through meiotic prophase (fig. S13B). Sequencing of the reverse transcription polymerase chain reaction (RT-PCR) products led us to revise the annotation of the mouse reference genome, yielding a protein of 579 amino acids and 63.8 kDa (fig. S14).

To investigate whether Top6bl has a role in meiotic DSB formation in mice, we generated point mutations in exon 9 of the Top6bl gene by means of CRISPR-Cas9 DSB targeting. Upon deletion or insertion, this led to a frameshift and a potential truncated protein of 279 amino acids without the transducer domain (18) (fig. S15). Male Top6bl−/− mice developed normally and reached adulthood but had smaller testes compared with wild-type (WT) mice (table S1). No phenotypic alteration could be detected in heterozygous Top6bl+/− mice. Histological analysis of Top6bl−/− testis sections showed that spermatogenesis was disrupted in most seminiferous tubules. Spermatogonia were present at the periphery of tubules like in WT mice; however, spermatocytes were strongly depleted and spermatids could not be detected, suggesting a defect during meiotic prophase (Fig. 4A).

Fig. 4 Top6bl is essential for spermatocyte and oocyte development.

(A) Periodic acid–Schiff staining of testis sections from WT (+/+) and Top6bl−/− (−/−) mice (n = 2 animals) at 30 dpp. (B) Hematoxylin and eosin staining of ovary sections from WT (+/+) (n = 1) and Top6bl−/− (−/−) (n = 4) mice at 30 dpp. PF, primordial follicles; PriF, primary follicles; GF, growing follicles. (C) Mean number with SD of primordial follicles, primary follicles, and growing follicles in Top6bl+/+ (n = 1) (gray bars), Top6bl+/− (n = 1) (white bars), and Top6bl−/− (n = 4) (black bars) ovaries at 30 dpp. Top6bl+/+ and Top6bl+/− were not significantly different (z test, P > 0.05), whereas Top6bl+/+ and Top6bl−/− were statistically different (z test, P < 0.0002).

To more precisely identify the meiotic defect, we monitored meiotic prophase by immunocytochemical analysis of spread spermatocytes using various markers to detect potential alterations in DSB formation or repair. Based on the phenotypes of Spo11 mutant mice (19, 20), defects in DSB formation have several consequences during prophase, particularly the reduction of phosphorylated H2AFX (γH2AFX) due to defective ATM kinase activation. The lack of DSBs also results in defective loading of DSB repair proteins (such as RPA and DMC1 that bind to single-stranded DNA present on processed DSB ends), as well as a defect in homologous synapsis (21). Top6bl−/− spermatocytes at leptotene- and zygotene-like stages showed reduced γH2AFX levels and undetectable RPA foci, consistent with a deficiency in DSB formation (Fig. 5A and fig. S16B). The nuclear localization of γH2AFX was also altered. In Top6bl−/− spermatocytes, γH2AFX did not accumulate specifically around the sex chromosomes but instead was enriched in subdomains (Fig. 5A) that could correspond to unsynapsed axes. Monitoring the loading of SYCP1, a component of the central element of the synaptonemal complex, indicated that synapsis formation was strongly altered in Top6bl−/− spermatocytes. Many chromosomes remained unsynapsed, and the axes containing synapsed stretches were often involved in interactions with several partners, suggesting interactions between nonhomologous chromosomes (Fig. 5B and fig. S16A). The presence of unsynapsed chromosomes is known to trigger the MSUC (meiotic silencing of unsynapsed chromatin) response, which leads to accumulation of γH2AFX on unsynapsed axes, even in the absence of meiotic DSB formation (22, 23), which is thus compatible with the detection of residual γH2AFX in Top6bl−/− spermatocytes. Similar defects in DSB and homologous synapsis formation were observed in a Top6bl−/− mutant generated independently (18) (founder 53614, figs. S15 and S17). To evaluate progression into meiotic prophase, we determined the expression of the testis-specific histone H1 variant (H1t) that is normally detected from mid-pachytene. Top6bl−/− spermatocytes were devoid of or showed only low H1t expression, indicating failure to progress through prophase and arrest before or at mid-pachytene (fig. S18). This finding also provides a possible explanation for the lack of late spermatocytes and later stages in testis sections (Fig. 4A). Notably, all phenotypes observed in Top6bl−/− spermatocytes are those predicted for a DSB formation defect and are identical to those reported in Spo11−/− mice (19,20). DSB formation also requires Mei1 and Mei4, which may act indirectly on Spo11 activity through unknown mechanisms (24). MEI4 localization on chromosome axes is Mei1-dependent and is correlated and required for efficient DSB activity (25, 26). As MEI4 localization was similar in Top6bl−/− and WT mice at leptotene (Fig. 5C), we could exclude the hypothesis that Top6bl indirectly affects DSB formation by altering MEI4 localization. Moreover, in Top6bl−/− spermatocytes at zygotene-like stages, MEI4 foci were persistent, as in Spo11−/− spermatocytes but unlike in WT spermatocytes, where they disappear upon DSB formation (26). MEI4 persistence in Top6bl−/− spermatocytes thus provides additional independent evidence for the lack of DSB formation. Finally, histological analysis of ovaries from Top6bl−/− females at 30 dpp showed that they were largely depleted of primordial and primary follicles compared with WT ovaries (Fig. 4, B and C). We therefore conclude that Top6bl is required for meiotic DSB formation during male and female meiosis.

Fig. 5 Top6bl is required for meiotic DSB formation.

(A) Immunocytochemical analysis with anti-γH2AFX, anti-SYCP3, and anti-RPA antibodies of spread spermatocytes from WT (+/+) and Top6bl−/− (−/−) mice (n = 2) at 30 dpp. Merged images of anti-SYCP3 and anti-RPA staining are shown. Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (B) Immunocytochemical analysis with anti-SYCP3 and anti-SYCP1 antibodies of spread spermatocytes from (+/+) and (−/−) mice (n = 2) at 30 dpp. Scale bars, 10 μm. (C) Immunocytochemical analysis with anti-SYCP3 and anti-MEI4 antibodies of spread spermatocytes from (+/+) and (−/−) mice (n = 2) at 30 dpp. Scale bars, 10 μm.

As evidenced by our study and the one performed on A. thaliana (10), the conserved structure and function of TOPOVIBL proteins demonstrate the notable evolutionary and biochemical relationship between the type IIB family of topoisomerases and meiotic DSB activity. On the basis of the phylogeny of several meiotic functions, it has become clear that sexual reproduction and meiosis have evolved very early during eukaryote evolution (27). As proposed for SPO11, the TOPOVIB family could also have expanded and diversified by gene duplication (28). Moreover, the presence of a TOPOVIBL subunit that interacts with SPO11 dramatically changes our view of how the biochemical step of DSB formation may work: In TopoVI, the B subunit is involved in strand passage and controlling dimer interfaces (29, 30). Although the involvement of a second DNA duplex is not expected for a SPO11-induced cleavage, the TOPOVIBL subunit may be involved in the regulation of DNA cleavage, through the direct interaction between the transducer domain and SPO11. The GHKL domain may be involved in the stabilization of the complex upon DNA cleavage, which could allow the reversibility of the reaction. However, SPO11 monomers may not need to be stably interacting upon DSB formation, and TOPOVIBL proteins that lack the GHKL domain, as observed in yeasts and Drosophila, may have lost the ability to maintain the complex upon DNA cleavage. As opposed to a TopoII-induced DNA cleavage, the challenge for SPO11-induced breaks is to be properly engaged in DSB repair by homologous recombination for maintenance of the stability of the genome, which also requires the coordinated involvement of several additional partners.

Correction (3 March 2016): Author affiliation 1 has been revised to include the University of Montpellier.

Supplementary Materials

www.sciencemag.org/content/351/6276/943/suppl/DC1

Materials and Methods

Figs. S1 to S18

Tables S1 and S2

References (3133)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: We thank M. Grelon for communicating information about the sequence of the A. thaliana MTOPVIB gene before publication, D. Gadelle and P. Forterre for numerous discussions and technical advice, all laboratory members for stimulating discussions and suggestions, M. A. Handel for the anti-H1t antibody, J. Cau from Montpellier RIO Imaging for image analysis, and the Cryopréservation Distribution Typage Archivage Animal Transgénése et Archivage d′Animaux Modéles and Réseau d′Histologie Expérimentale de Montpellier facilities for services. H.-M.B. was funded by grants from CNRS. B.d.M. was funded by grants from CNRS and the European Research Council Executive Agency under the European Community’s Seventh Framework Programme [FP7/2007-2013 grant agreement no. 322788]. T.R., H.-M.B., and B.d.M. supervised the project and wrote the manuscript. T.R., H.-M.B, A.N., C.M., B.C., and C.B. performed experiments.
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