Biology subjects

Keeney, S.

Publications and source records attributed to Keeney, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2020

De novo deletion mutations at recombination hotspots in mouse germlines

Numerous DNA double-strand breaks (DSBs) arise during meiosis to initiate homologous recombination. These DSBs are usually repaired faithfully, but here we uncover a new type of mutational event in which deletions form via joining of ends from two closely spaced DSBs (double cuts) within a single hotspot or at adjacent hotspots on the same or different chromatids. Deletions occur in normal meiosis but are much more frequent when DSB formation is dysregulated in the absence of the ATM kinase. Events between chromosome homologs point to multi-chromatid damage and aborted gap repair. Some deletions contain DNA from other hotspots, indicating that double cutting at distant sites creates substrates for insertional mutagenesis. End joining at double cuts can also yield tandem duplications or extrachromosomal circles. Our findings highlight the importance of DSB regulation and reveal a previously hidden potential for meiotic mutagenesis that is likely to affect human health and genome evolution.

molecular biology↗

bioRxiv2020

Deterioration of Glutaraldehyde Crosslinked Heterograft Biomaterials due to Advanced Glycation End Product Formation and Serum Albumin Infiltration

Bioprosthetic heart valves (BHV) are fabricated from glutaraldehyde cross-linked heterograft tissue, such as bovine pericardium (BP) or porcine aortic valves. BHV develop structural valve degeneration (SVD), often with calcification, requiring BHV replacement. Advanced glycation end products (AGE) are post-translational, non-enzymatic carbohydrate protein modifications. AGE are present in SVD-BHV clinical explants and not detectable in unimplanted BHV. Here, we studied the hypothesis that BHV susceptibility to AGE formation and serum protein infiltration results in deterioration of both leaflet collagen structure and mechanical properties. In vitro experiments studied BP and porcine collagen sponges (CS) for susceptibility to AGE formation using 14C-glucose and 14C-glyoxal with and without bovine serum albumin (BSA), as a model serum protein. The results showed AGE formation is a rapid and progressive process. BSA co-incubations reduced glyoxal and glucose uptake by BP and CS. Incubating BP in BSA caused a substantial increase in BP mass, enhanced by glyoxal co-incubation. Per two-photon microscopy, BP with AGE formation and BSA infiltration each induced significant disruption in collagen microarchitecture, with loss of collagen alignment and crimp. These effects are cumulative with the greatest disruption occurring when there was both AGE formation and BSA infiltration. Uniaxial testing of CS demonstrated that AGE formation, together with BSA uptake compared to controls, caused a significant deterioration in mechanical properties with a loss of viscoelastic relaxation and increased stiffness. It is concluded that AGE-BSA associated collagen structural disruption and deterioration of mechanical properties contribute to SVD.

biophysics↗

bioRxiv2020

Chromosome-autonomous feedback downregulates meiotic DSB competence upon synaptonemal complex formation

The number of DNA double-strand breaks (DSBs) initiating meiotic recombination is elevated in Saccharomyces cerevisiae mutants that are globally defective in forming crossovers and synaptonemal complex (SC), a protein scaffold juxtaposing homologous chromosomes. These mutants thus appear to lack a negative feedback loop that inhibits DSB formation when homologs engage one another. This feedback is predicted to be chromosome autonomous, but this has not been tested. Moreover, what chromosomal process is recognized as "homolog engagement" remains unclear. To address these questions, we evaluated effects of homolog engagement defects restricted to small portions of the genome using karyotypically abnormal yeast strains with a homeologous chromosome V pair, monosomic V, or trisomy XV. We found that homolog-engagement-defective chromosomes incurred more DSBs, concomitant with prolonged retention of the DSB-promoting protein Rec114, while the rest of the genome remained unaffected. SC-deficient, crossover-proficient mutants ecm11 and gmc2 experienced increased DSB numbers diagnostic of homolog engagement defects. These findings support the hypothesis that SC formation provokes DSB protein dissociation, leading in turn to loss of a DSB competent state. Our findings show that DSB number is regulated in a chromosome-autonomous fashion and provide insight into how homeostatic DSB controls respond to aneuploidy during meiosis. bioRxiv version 2 (September 2020) We added one bioRxiv citation in the discussion section which shows increased breaks in gmc2 and ecm11 mutants. This work was done independently by Keun Kim, Miki Shinohara and colleagues. The pulse gel electrophoresis result shown in Figure 1D has been re-quantified. The conclusion remains that homeologous and monosomic chromosome V generate more DSBs compared to internally controlled chromosome III. One zip3 map included in PCA (Figure 6C) and hierarchical clustering analysis (Figure S5B) has been deleted because this particular map has not yet been published. This change does not affect the conclusion. An additional supplemental table (S5) has been added to show Spo11-oligo datasets used in this study. Figures and preprint have been reformatted. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/089367v2_fig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@871eaforg.highwire.dtl.DTLVardef@ef7995org.highwire.dtl.DTLVardef@176441org.highwire.dtl.DTLVardef@1b6e772_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Higher DSB levels on homolog-engagement defective chromosomes. (A) Cartoons of wild-type, homeologous, monosomic and trisomic chromosome configurations. Grey lines are S. cerevisiae chromosomes and orange lines are S. pastorianus. The homeologous chrV pair rarely synapses or recombines. The trisomic chromosomes can adopt different synaptic configurations ("II+I", partner switch, and triple synapsis). (B) Representative PFGE Southern blots probed for chrIII and chrV. P, signal from parental-length DNA; W, signal in wells. Asterisk indicates an ectopic recombination product between leu2::hisG (on chrIII) and ho::hisG (on chrIV) in the monosomic strain. The other strains do not form this product because they do not have a hisG insert at ho. (C) Poisson-corrected DSB quantification of PFGE Southern blots. (D) Quantification of time-averaged DSBs on chrV relative to chrIII for wild-type, homeologous and monosomic strains. * p<0.05, unpaired t test. Error bars in C, D are mean {+/-} SD except for the monosomic strain (mean {+/-} range). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/089367v2_fig6.gif" ALT="Figure 6"> View larger version (36K): org.highwire.dtl.DTLVardef@1152edforg.highwire.dtl.DTLVardef@1b853daorg.highwire.dtl.DTLVardef@89392dorg.highwire.dtl.DTLVardef@108d65a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO gmc2{Delta} and ecm11{Delta} mutants share similar homolog-engagement signatures as zip3{Delta}. (A, B) Representative labeling of Spo11-oligo complexes in gmc2{Delta} (A) and ecm11{Delta} (B) mutants and quantification relative to wild-type cultures processed in parallel. Error bars indicate mean {+/-} SD for 3 cultures. Radiolabeled Spo11-oligo complexes are detected by autoradiography (left panel, top) and total Spo11 is detected by anti-Flag western blot (WB; left panel, bottom). The two main labeled species differ in the sizes of oligos (Neale et al. 2005). Most Spo11 protein does not end up making DSBs, so nearly all of the visible western blot signal is from free Spo11 that does not have an oligo attached (Neale et al. 2005). (C) Principal component analysis of 21 wild-type and mutant Spo11-oligo maps. (D) Loss of anticorrelation between chromosome length and DSB density in homolog-engagement-defective mutants. Each point is one chromosome. Correlation coefficients (Pearsons r) are shown. (E) Fold change of Spo11-oligo counts in different chromosomal domains. Tel, within 20 kb of telomeres; Cen, within 10 kb of centromeres; rDNA, from 60 kb leftward to 30 kb rightward of rDNA; Interstitial, all others. Horizontal dashed lines mark values assumed as no change (black) and average change (1.8-fold for zip3{Delta}, cyan; 1.7-fold for ecm11{Delta}, purple). Boxes indicate median and interquartile range; whiskers indicate the most extreme data points that are [≤] 1.5 times the interquartile range from the box; individual points are outliers C_FIG O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/089367v2_figS5.gif" ALT="Figure 5"> View larger version (35K): org.highwire.dtl.DTLVardef@130cbf6org.highwire.dtl.DTLVardef@9ebe8forg.highwire.dtl.DTLVardef@1f0a2b4org.highwire.dtl.DTLVardef@14a0fcf_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOSupplemental Figure S5.C_FLOATNO DSB patterns in gmc2{Delta} and ecm11{Delta} mutants. (A) Meiotic progression showing percentage of cells completing the first divisions (total bi- and tetra-nucleate cells, mean {+/-} SD, 3 cultures each). At least 100 cells were counted at each time point for each culture. (B) Hierarchical clustering analysis using the "Ward D2" method for 21 wild-type and mutant maps. The y-axis represents height of the cluster dendrogram. In the label for each data set, the letters A, B, C and D stand for maps generated by Xiaojing Mu, Neeman Mohibullah, Megan van Overbeek, and Xuan Zhu, respectively, and the numerals distinguish biological replicate maps. (C) Regional variation in response to zip3{Delta} and ecm11{Delta} mutations for chromosome XII. Lines, local regression (loess) based on Spo11-oligo density changes in 5 kb-bins; green circle, centromere; rDNA location is labeled. (D, E) Fold change of Spo11-oligo densities for zip3{Delta} and ecm11{Delta} compared to wild type in pericentric (D) and telomere-proximal (E) regions. Lines, local regression (loess) for data in 5 kb-bins averaged across 32 chromosome arms; dashed line, no change in black and genome average change in cyan (1.8-fold, zip3{Delta}) and purple (1.7-fold, ecm11{Delta}); yellow shading, zones suppressed for DSB formation as previously defined (Pan etal. 2011). C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@15408baorg.highwire.dtl.DTLVardef@9171aorg.highwire.dtl.DTLVardef@cbabfaorg.highwire.dtl.DTLVardef@187d594org.highwire.dtl.DTLVardef@d6eb68_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOSupplemental Table S5.C_FLOATNO O_TABLECAPTIONSpo11-oligo datasets used in this study C_TABLECAPTION C_TBL

genetics↗

bioRxiv2020

Structural and functional characterization of the Spo11 core complex

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.

molecular biology↗

bioRxiv2020

DNA-dependent macromolecular condensation drives self-assembly of the meiotic DNA break machinery

Formation of meiotic DNA double-strand breaks (DSBs) by Spo11 is tightly regulated and tied to chromosome structure, but the higher-order assemblies that execute and control DNA breakage are poorly understood. We address this question through molecular characterization of Saccharomyces cerevisiae RMM proteins (Rec114, Mei4 and Mer2)--essential, conserved components of the DSB machinery. Each subcomplex of Rec114-Mei4 (2:1 heterotrimer) or Mer2 (homotetrameric coiled coil) is monodisperse in solution, but they independently condense with DNA into dynamic, reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive condensation, which correlates with DSB formation in vivo. Condensates fuse into mixed Rec114-Mei4-Mer2 clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centers of DSB activity. We propose that multilayered control of Spo11 arises from recruitment of regulatory components and modulation of biophysical properties of the condensates.

molecular biology↗

bioRxiv2020

Glycation and Serum Albumin Infiltration Contribute to the Structural Degeneration of Bioprosthetic Heart Valves

BackgroundBioprosthetic heart valves (BHV) are widely used to treat heart valve disease but are fundamentally limited by structural valve degeneration (SVD). Non-calcific mechanisms of SVD entirely account for approximately 30% of SVD cases and contribute to calcific SVD but remain understudied. Glycation mechanisms have not been previously associated with SVD, despite being established as degenerative in collagenous native tissues. ObjectivesTo determine whether blood component infiltration-based glycation and concomitant human serum albumin (HSA) deposition contribute mechanistically to SVD. MethodsImmunohistochemistry (IHC) was used to identify advanced glycation end-products (AGEs) and serum albumin accumulation in 45 aortic valve BHV explanted due to SVD, glutaraldehyde-treated bovine pericardium (BP) incubated in vitro in glyoxal and HSA, and rat subcutaneous BP implants. Structural impacts of glycation-related mechanisms were evaluated by second harmonic generation (SHG) collagen imaging. Hydrodynamic effects of valve glycation and concomitant HSA exposure were studied with an ISO-5840-compliant pulse duplicator system using surgical grade BHV. ResultsAll 45 clinical explants and in vitro-incubated BP demonstrated accumulated AGE and HSA compared to un-implanted, un-exposed BHV. SHG revealed instigation of collagen malalignment similar to that in SVD explants by glycation and HSA infiltration. Rat subdermal explants also showed AGE and serum albumin accumulation. Pulse duplication demonstrated significantly reduced orifice area and increased pressure gradient and peak fluid velocity following glyoxal and HSA incubations. ConclusionsGlycation and concomitant HSA infiltration occur in clinical BHV and contribute to structural and functional degeneration of leaflet tissue, thus representing novel, interacting mechanisms of BHV SVD.

bioengineering↗

bioRxiv2020

shani mutation in mouse affects splicing of Spata22 and leads to impaired meiotic recombination

Recombination is crucial for chromosome pairing and segregation during meiosis. SPATA22, along with its direct binding partner and functional collaborator, MEIOB, is essential for the proper repair of double-strand breaks (DSBs) during meiotic recombination. Here we describe a novel point-mutated allele (shani) of mouse Spata22 that we isolated in a forward genetic screen. shani mutant mice phenocopy Spata22-null and Meiob-null mice: mutant cells appear to form DSBs and initiate meiotic recombination, but are unable to complete DSB repair, leading to meiotic prophase arrest, apoptosis and sterility. shani mutants show precocious loss of DMC1 foci and improper accumulation of BLM-positive recombination foci, reinforcing the requirement of SPATA22-MEIOB for the proper progression of meiotic recombination events. The shani mutation lies within a Spata22 coding exon and molecular characterization shows that it leads to incorrect splicing of the Spata22 mRNA, ultimately resulting in no detectable SPATA22 protein. We propose that the shani mutation alters an exonic splicing enhancer element (ESE) within the Spata22 transcript. The affected DNA nucleotide is conserved in most tetrapods examined, suggesting that the splicing regulation we describe here may be a conserved feature of Spata22 regulation.

genetics↗

bioRxiv2019

Concerted cutting by Spo11 illuminates DNA break mechanisms and initiates gap repair during meiosis

Genetic variation arises during meiosis via repair of DNA double-strand breaks (DSBs) created by the topoisomerase-like Spo11 protein. These DSBs are thought to always occur sparsely across the genome, with isolated DSBs generating discrete recombination events. We challenge this view, demonstrating that hyper-localised coincident DSBs frequently form within hotspots in both S. cerevisiae and mouse--a process suppressed by the DNA damage response kinase Tel1/ATM. Remarkably, the distances separating coincident DSBs vary with [~]10.5 bp periodicity, invoking a model where adjacent Spo11 molecules have a fixed orientation relative to the DNA helix. Deep sequencing of meiotic progeny identifies recombination scars consistent with gap repair initiated by adjacent DSBs. Our results revise current thinking about how genetic recombination initiates, reviving original concepts of meiotic recombination as double-strand gap repair.

molecular biology↗

bioRxiv2019

Molecular structures and mechanisms of DNA break processing in mouse meiosis

Exonucleolytic resection, critical to repair double-strand breaks (DSBs) by recombination, is not well understood, particularly in mammalian meiosis. Here, we define structures of resected DSBs in mouse spermatocytes genome-wide at nucleotide resolution. Resection tracts averaged 1100 nucleotides, but with substantial fine-scale heterogeneity at individual hotspots. Surprisingly, EXO1 is not the major 5'[->]3' exonuclease, but the DSB-responsive kinase ATM proved a key regulator of both initiation and extension of resection. In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions, consistent with joint molecules with incompletely invaded 3' ends. Finally, we provide evidence for PRDM9-dependent chromatin remodeling leading to increased accessibility at recombination sites. Our findings give insight into the mechanisms of DSB processing and repair in meiotic chromatin.

molecular biology↗

bioRxiv2019

Mechanism of in vivo activation of the MutLγ-Exo1 complex for meiotic crossover formation

Crossovers generated during the repair of programmed double-strand breaks (DSBs) during homologous recombination are essential for fertility to allow accurate homolog segregation during the first meiotic division. Most crossovers arise through the cleavage of recombination intermediates by the Mlh1-Mlh3 (MutL{gamma}) endonuclease and an elusive non-catalytic function of Exo1, and require the Polo kinase Cdc5. Here we show in budding yeast that MutL{gamma} forms a constitutive complex with Exo1, and in meiotic cells transiently contacts the Msh4-Msh5 (MutS{gamma}) heterodimer, also required for crossover formation. We further show that MutL{gamma}-Exo1 associates with recombination intermediates once they are committed to the crossover repair pathway, and then Exo1 recruits Cdc5 through a direct interaction that is required for activating MutL{gamma} and crossover formation. Exo1 therefore serves as a non-catalytic matchmaker between Cdc5 and MutL{gamma}. We finally show that in vivo, MutL{gamma} associates with the vast majority of DSB hotspots, but at a lower frequency near centromeres, consistent with a strategy to reduce at-risk crossover events in these regions. Our data highlight the tight temporal and spatial control of the activity of a constitutive, potentially harmful, nuclease.

molecular biology↗

bioRxiv2019

Mechanistic insight into crossing over during mouse meiosis

Characteristics of heteroduplex DNA illuminate how strands exchange during homologous recombination, but mismatch correction can obscure them. To investigate recombination mechanisms, meiotic crossover products were analyzed at two hotspots in Msh2-/- mice containing homologous chromosomes derived from inbred strains. Recombination frequencies were unchanged in the mutant, implying that MSH2-dependent recombination suppression does not occur at this level of diversity. However, a substantial fraction of crossover products retained heteroduplex DNA in the absence of MSH2, and some also had multiple switches between parental markers suggestive of MSH2-independent correction. Recombinants appeared to reflect a biased orientation of crossover resolution, possibly stemming from asymmetry at DNA ends established in earlier intermediates. Many crossover products showed no evidence of heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these two modifications of the original double-strand break repair model may be sufficient to explain most meiotic crossing over in mice.

genetics↗

bioRxiv2019

Cyclin B3 is dispensable for mouse spermatogenesis

Cyclins, as regulatory partners of cyclin-dependent kinases (CDKs), control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation of genomes. Compared to mitosis, relatively little is known about how cyclin-CDK complexes control meiosis, the specialized cell division that generates gametes for sexual production. Mouse cyclin B3 was previously shown to have expression restricted to the beginning of meiosis, making it a candidate to regulate meiotic events. Indeed, female mice lacking cyclin B3 are sterile because oocytes arrest at the metaphase-to-anaphase transition of meiosis I. However, whether cyclin B3 functions during spermatogenesis was untested. Here, we found that males lacking cyclin B3 are fertile and show no detectable defects in spermatogenesis based on histological analysis of seminiferous tubules. Cytological analysis further showed no detectable defects in homologous chromosome synapsis or meiotic progression, and suggested that recombination is initiated and completed efficiently. Moreover, absence of cyclin B3 did not exacerbate previously described meiotic defects in mutants deficient for cyclin E2, suggesting a lack of redundancy between these cyclins. Thus, unlike in females, cyclin B3 is not essential for meiosis in males despite its prominent meiosis-specific expression.

genetics↗

bioRxiv2019

Ensuring meiotic DNA break formation in the mouse pseudoautosomal region

Sex chromosomes in males share only a diminutive homologous segment, the pseudoautosomal region (PAR), wherein meiotic double-strand breaks (DSBs), pairing, and crossing over must occur for correct segregation. How cells ensure PAR recombination is unknown. Here we delineate cis-and trans-acting factors that control PAR ultrastructure and make the PAR the hottest area of DSB formation in the male mouse genome. Prior to DSB formation, PAR chromosome axes elongate, sister chromatids separate, and DSB-promoting factors hyperaccumulate. These phenomena are linked to mo-2 minisatellite arrays and require ANKRD31 protein. We propose that the repetitive PAR sequence confers unique chromatin and higher order structures crucial for DSB formation, X-Y pairing, and recombination. Our findings establish a mechanistic paradigm of mammalian sex chromosome segregation during spermatogenesis.

molecular biology↗

bioRxiv2018

REC114 partner ANKRD31 controls number, timing and location of meiotic DNA breaks

Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation. Here we unveil mouse ANKRD31 as a lynchpin governing multiple aspects of DSB formation. Spermatocytes lacking ANKRD31 have altered DSB locations and fail to target DSBs to sex chromosomes pseudoautosomal regions (PAR). They also have delayed/fewer recombination sites but, paradoxically, more DSBs, suggesting DSB dysregulation. Unrepaired DSBs and pairing failures--stochastic on autosomes, nearly absolute on X and Y--cause meiotic arrest and sterility in males. Ankrd31-deficient females have reduced oocyte reserves. A crystal structure defines direct ANKRD31- REC114 molecular contacts and reveals a surprising pleckstrin homology domain in REC114. In vivo, ANKRD31 stabilizes REC114 association with the PAR and elsewhere. Our findings inform a model that ANKRD31 is a scaffold anchoring REC114 and other factors to specific genomic locations, promoting efficient and timely DSB formation but possibly also suppressing formation of clustered DSBs.

genetics↗

bioRxiv2018

Multilayered mechanisms ensure that short chromosomes recombine in meiosis

To segregate accurately during meiosis, homologous chromosomes in most species must recombine. Very small chromosomes would risk missegregation if recombination were randomly distributed, so the double-strand breaks (DSBs) that initiate recombination are not haphazard. How this nonrandomness is controlled is not under-stood. Here we demonstrate that Saccharomyces cerevisiae integrates multiple, temporally distinct pathways to regulate chromosomal binding of pro-DSB factors Rec114 and Mer2, thereby controlling duration of a DSB-competent state. Homologous chromosome engagement regulates Rec114/Mer2 dissociation late in prophase, whereas replication timing and proximity to centromeres or telomeres influence timing and amount of Rec114/Mer2 accumulation early. A distinct early mechanism boosts Rec114/Mer2 binding quickly to high levels specifically on the shortest chromosomes, dependent on chromosome axis proteins and subject to selection pressure to maintain hyperrecombinogenic properties of these chromosomes. Thus, an organisms karyotype and its attendant risk of meiotic missegregation influence the shape and evolution of its recombination landscape.

genetics↗

bioRxiv2018

Cyclin B3 promotes APC/C activation and anaphase I onset in oocyte meiosis

As obligate kinase partners, cyclins control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation of genomes. Meiosis, the cell division that generates gametes for sexual reproduction, poses unique challenges because two rounds of chromosome segregation must be executed without intervening DNA replication. Mammalian cells express numerous, temporally regulated cyclins, but how these proteins collaborate to control meiosis remains poorly understood. Here, we delineate an essential function for mouse cyclin B3 in the first meiotic division of oocytes. Females genetically ablated for cyclin B3 are viable, indicating the protein is dispensable for mitotic divisions, but are sterile. Mutant oocytes appear normal until metaphase I but then display a highly penetrant failure to transition to anaphase I. They arrest with hallmarks of defective APC/C activation, including no separase activity and high MPF, cyclin B1, and securin levels. Partial APC/C activation occurs, however, as exogenously expressed APC/C substrates can be degraded and arrest can be suppressed by inhibiting MPF kinase. Cyclin B3 is itself targeted for degradation by the APC/C. Cyclin B3 forms active kinase complexes with CDK1, and meiotic progression requires cyclin B3-associated kinase activity. Collectively, our findings indicate that cyclin B3 is essential for oocyte meiosis because it fine-tunes APC/C activity as a kinase-activating CDK partner. Cyclin B3 homologs from frog, zebrafish, and fruitfly rescue meiotic progression in cyclin B3-deficient mouse oocytes, indicating conservation of the biochemical properties and possibly cellular functions of this germline-critical cyclin.

cell biology↗

bioRxiv2018

MEIOB and SPATA22 resemble RPA subunits and interact with the RPA complex to promote meiotic recombination

Homologous recombination is a conserved DNA repair process mandatory for chromosome segregation during meiosis. RPA, a ubiquitous complex essential to recombination, is thought to play a similar role during mitotic and meiotic recombination. MEIOB, a meiosis-specific factor with unknown molecular function, ressembles a RPA subunit. Here we use in vivo approaches to show that in mouse spermatocytes, DMC1 and RAD51 appear to be normally loaded in the absence of MEIOB but are prematurely lost from unrepaired recombination sites. This loss correlates with an accumulation of the BLM helicase on meiotic chromosomes. We also show that MEIOB alters the immunodetection of RPA subunits at meiotic recombination sites. Using electron microscopy and purified proteins, we demonstrate that the MEIOB-SPATA22 complex associates with and modifies the conformation of RPA-coated ssDNA. Finally, we identify structural homology between MEIOB, SPATA22 and RPA subunits, and show that MEIOB and SPATA22 interact through C-terminal OB-fold containing domains (OBCDs) like RPA subunits. Moreover, MEIOB and SPATA22 cooperate to interact with RPA through their OBCDs. Our results suggest that MEIOB, SPATA22 and RPA work together to ensure proper processing of meiotic recombination intermediates.

cell biology↗

bioRxiv2017

Shu complex SWS1-SWSAP1 is required for mouse meiotic recombination in concert with the BRCA2 C terminus

Homology recognition and DNA-strand invasion ensure faithful homolog pairing and segregation during the first meiotic division1. RAD51 and DMC1 recombinases catalyze these steps2, with BRCA2 promoting their assembly into nuclear foci3. The recently identified human SWS1-SWSAP1 complex, related to the Shu complex in yeast, promotes RAD51 focus formation in cell lines4,5. We show here that mouse SWS1-SWSAP1 is critical for meiotic homologous recombination (HR) by promoting the assembly of RAD51 and DMC1 on early recombination intermediates. Absence of the complex perturbs meiotic progression in males and females and both sexes are sterile, although a fraction of meiocytes form crossovers. Remarkably, loss of the DNA damage checkpoint kinase CHK2 rescues fertility specifically in females without rescuing crossover numbers. Unlike the Shu complex, the BRCA2 C terminus (known to be required for RAD51 stabilization6,7) is dispensible for RAD51 and DMC1 focus formation. However, concomitant loss of the BRCA2 C terminus aggravates the meiotic defects in Shu mutant spermatocytes. These results point to a complex interplay of factors that ensure recombinase function and hence meiotic progression in the mouse.

genetics↗