MEILB2-BRME1 forms a V-shaped DNA clamp upon BRCA2-binding in meiotic recombination

DNA double-strand break repair by homologous recombination has a specialised role in meiosis by generating crossovers that enable the formation of haploid germ cells. This requires meiosis-specific MEILB2-BRME1, which interacts with BRCA2 to facilitate loading of recombinases onto resected DNA ends. Here, we report the crystal structure of the MEILB2-BRME1 2:2 core complex, revealing a parallel four-helical assembly that recruits BRME1 to meiotic double-strand breaks in vivo. It forms an N-terminal β-cap that binds to DNA, and a MEILB2 coiled-coil that bridges to C-terminal ARM domains. Upon BRCA2-binding, MEILB2-BRME1 2:2 complexes dimerize into a V-shaped 2:4:4 complex, with rod-like MEILB2-BRME1 components arranged at right-angles. The β-caps located at the tips of the MEILB2-BRME1 limbs are separated by 25 nm, allowing them to bridge between DNA molecules. Thus, we propose that BRCA2 induces MEILB2-BRME1 to function as a DNA clamp, connecting resected DNA ends or homologous chromosomes to facilitate meiotic recombination.


Introduction
Breast cancer susceptibility protein BRCA2 performs a central role in recombination-mediated DNA double-strand break (DSBs) repair by loading recombinases onto resected DNA ends (Jensen et al., 2010, Liu et al., 2010, Thorslund et al., 2010. Its importance in genome stability is demonstrated by the gross chromosomal rearrangements that accumulate in BRCA2 deficient mammalian cells (Patel et al., 1998, Yu et al., 2000, and the strong association between germline BRCA2 mutations and early-onset breast and ovarian cancers (Hall et al., 1990). BRCA2 is also required for meiosis, as its germline-specific deficiency leads to meiotic impairment and infertility in mice (Sharan et al., 2004).
Here, programmed DSBs are induced, and then repaired by recombination between homologous chromosomes to generate crossovers, which provide genetic diversity and are critical for the correct segregation of homologues (Hunter, 2015, Zickler andKleckner, 2015). Hence, BRCA2 is essential for maintaining genome integrity in somatic cells and for generating haploid germ cells in meiosis.
BRCA2 is a large protein (3,329 amino-acids in mice) that contains multiple recombinase-binding sites and a single globular DNA-binding domain (Figure 2a). BRC repeats within the central exon 11 region bind to RAD51 and DMC1 by competing with their oligomerisation (Pellegrini et al., 2002, Martinez et al., 2016. In contrast, sequences that flank the DNA-binding domain (exons 14 and 27), interact with DMC1 and RAD51, respectively, in a manner that stabilises their nucleoprotein filaments (Davies and Pellegrini, 2007, Thorslund et al., 2007. Finally, the DNAbinding domain contains OB-folds for ssDNA-binding (Yang et al., 2002), and interacts with acidic DNA-mimicking protein DSS1 (Marston et al., 1999), which act together to offload RPA from ssDNA overhangs (Zhao et al., 2015, Bell et al., 2023. Hence, whilst we lack a full molecular mechanism, BRCA2 contains the necessary functionalities to explain its role in displacing RPA, and loading recombinases onto ssDNA overhangs to form nucleoprotein filaments (Jensen et al., 2010, Liu et al., 2010, Thorslund et al., 2010.
In MEILB2 and BRME1 knockout mice, males are sterile, with failure of RAD51 and DMC1 foci formation, failure of DSB repair and failure of crossover formation (Zhang et al., 2019, Brandsma et al., 2019, Felipe-Medina et al., 2020, Takemoto et al., 2020, Shang et al., 2020. In contrast, females exhibit subfertility, in which the development and number of oocytes is substantially reduced (Zhang et al., 2019, Brandsma et al., 2019, Felipe-Medina et al., 2020, Takemoto et al., 2020, Shang et al., 2020, Shang et al., 2022. This resembles the sexual dimorphism observed upon deficiency of BRCA2 and other recombination factors (Sharan et al., 2004). Their co-localisation and similar phenotypes suggest that MEILB2 and BRME1 exist within the same functional unit in meiosis. This likely occurs through modulation of BRCA2 function as MEILB2 or BRME1 expression suppresses BRCA2-mediated recombination in somatic cells (Sato et al., 2020, and MEILB2 expression has been observed in cancer cell lines and human tumour samples (Brandsma et al., 2019, Sato et al., 2020. Indeed, it has been hypothesised that MEILB2-BRME1 may module the oligomeric state or conformation of BRCA2 to favour meiotic rather than mitotic recombination .
The mechanical basis of recombination is an intrinsically structural problem that will likely be solved by understanding the functional architecture of its principal components and complexes. MEILB2 consists of N-terminal coiled-coil and C-terminal ARM (armadillo repeat) domains, which bind with high affinity to BRME1 and BRCA2, respectively , Pendlebury et al., 2021 (Figure   2a). The ARM domains interact with the MBD (MEILB2-binding domain) of BRCA2, which is located immediately upstream of its DMC1-binding site   (Figure 2a). Crystal structures have revealed that BRCA2-binding staples together two MEILB2-ARM dimers at approximately 90° to one another (Pendlebury et al., 2021, Ghouil et al., 2021. In these 2:4 complexes, each BRCA2 MBD peptide bridges between interacting MEILB2 ARM dimers, through cryptic repeats within their Nand C-terminal ends binding to opposing ARM domains (Pendlebury et al., 2021, Ghouil et al., 2021.
The N-terminus of MEILB2's coiled-coil binds to the C-terminal end of BRME1 , which currently has no other known structure or interacting partners. The MEILB2 coiled-coil exists in dimeric and higher-order species in isolation, but forms stable 2:2 complexes with BRME1 . However, the lack of structural information regarding the MEILB2-BRME1 complex currently prevents us from understanding the architecture of the wider BRCA2-MEILB2-BRME1 assembly, and critically the molecular basis of MEILB2-BRME1 function in BRCA2-mediated meiotic recombination.
Here, we report the crystal structure of the MEILB2-BRME1 2:2 core complex, revealing a four-helical assembly that mediates BRME1 recruitment to meiotic DSBs in vivo. We show that its N-terminal cap binds to DNA, requiring both MEILB2 and BRME1 amino-acids. We report a model of the full MEILB2-BRME1 2:2 complex, and show that BRCA2-binding induces dimerization of this structure into a V-shaped assembly. The -caps at the ends of the MEILB2-BRME1 limbs of this structure are separated by 25 nm, so bind together distinct DNA molecules, forming protein-DNA networks. Hence BRCA2-binding can induce MEILB2-BRME1 to act as a DNA clamp in meiotic recombination.

Crystal structure of MEILB2-BRME1
MEILB2 possesses an N-terminal -helical region, consisting of two predicted -helices (1 and 2), followed by a C-terminal ARM domain (Figure 2a). We previously demonstrated that MEILB2's helical region binds via its 1 helix to the C-terminus of BRME1 (MEILB2-binding domain, MBD), forming a stable 2:2 complex . Here, we identified an optimised construct for crystallography, which includes the α1 helix and the beginning of the α2 helix of MEILB2 (aminoacids 22-81; herein referred to as MEILB2 core) and the core of BRME1's MBD (amino-acids 540-578; herein referred to as BRME1) (Supplementary Figure 1). Crystals of this MEILB2-BRME1 core complex diffracted to a resolution limit of 1.50 Å, enabling structure solution by molecular replacement of ideal helical fragments using ARCIMBOLDO_LITE (Caballero et al., 2018) (Table 1 and Supplementary   Figure 2). This revealed a 2:2 structure, consisting of an N-terminal parallel four-helical bundle of MEILB2 1 and BRME1, followed by a C-terminal MEILB2 2 parallel dimeric coiled-coil ( Figure 2b).
The MEILB2-BRME1 core structure contains an unusual -cap at its N-terminal tip (Figure 2b,c). This consists of an anti-parallel two-stranded -sheet, which is formed by the N-terminal ends of MEILB2 chains, and is orientated perpendicular to the helical axis (Figure 2b,c). The -cap binds together the N-termini of the two 1 helices of the four-helical bundle, so likely prevents the fraying apart of helical ends that typically occurs within coiled-coil structures (Dragan and Privalov, 2002). Hence, we infer that the -cap likely confers rigidity to the N-terminal end of the MEILB2-BRME1 2:2 structure.
In previous cases of /-coiled-coils, the -sheets typically constitute insertions, as -layers within the -helical coiled-coil structure (Hartmann et al., 2016). To our knowledge, MEILB2-BRME1 is the first example of an /-coiled-coil in which the -sheet caps off the end of the -helical coiled-coil.
Following the β-cap, a MEILB2-BRME1 four-helical bundle is observed, terminating with the splaying apart of the C-termini of BRME1 chains (Figure 2b,d). At this point, there is slight 'kink' in the MEILB2 chains, between 1 and2 helices, where L50 and N51 adopt non-helical conformations due to a single amino-acid insertion into the heptad repeats ( Figure 2e). This kink re-orientates the MEILB2 chains from the upstream four-helical bundle to the downstream dimeric coiled-coil conformation ( Figure 2b,e). The parallel dimeric coiled-coil of the α2 helix adopts a canonical heptad pattern ( Figure 2b,f). Thus, the overall structure of the MEILB2-BRME1 core can be described as a parallel 2:2 α/β-coiled-coil, where an N-terminal MEILB2 β-cap is followed by a MEILB2 α1-BRME1 four-helical bundle that transitions via a kink into a C-terminal MEILB2 α2 dimeric coiled-coil (Figure 2b,g).

BRME1 recruitment to meiotic DSBs requires its MEILB2-binding interface
To determine whether the observed MEILB2-BRME1 interaction in the crystal structure is crucial for their interaction in vivo, we generated a point mutant of BRME1 that specifically targets its MEILB2binding site. The hydrophobic core of the four-helical bundle comprises highly conserved BRME1 amino acids V548, L555, and I562, which occupy the 'a' positions within the heptad repeats ( Figure   3a). Therefore, we introduced glutamate mutations at these residues (V548E, L555E, and I562E; hereafter referred to as 3E) to disrupt the assembly of the four-helical bundle. We confirmed this disruption biochemically, demonstrating that the 3E mutation effectively prevented the formation of In a previous study, we established that GFP-BRME1 is recruited to meiotic DSBs upon expression in mouse spermatocytes through in vivo electroporation . Therefore, we employed the same system to investigate the impact of the 3E mutation on the localization of full-length BRME1. While GFP-BRME1 FL 3E was expressed at a comparable level to the wild-type protein ( Figure   3c), it failed to be recruited to meiotic DSBs in zygotene and pachytene spermatocytes ( Figure 3d).
These findings confirm that the MEILB2-binding interface of BRME1 observed in the crystal structure is solely responsible for its interaction with MEILB2 and its recruitment to meiotic DSBs in vivo.

Structure of the full MEILB2-BRME1 complex
Our MEILB2-BRME1 core crystal structure contains the N-terminal end of the MEILB2 2 coiled-coil, up to amino-acid Q77 (Figure 2b). The C-terminal end of the same coiled-coil, from the equivalent of amino-acid Q109, was observed in previous crystal structures of the BRCA2-MEILB2 ARM complex (PDB accessions 7LDG and 7BDX; Pendlebury et al., 2021, Ghouil et al., 2021. Hence, MEILB2 2 likely forms a continuous coiled-coil that physically separates the BRME1-and BRCA2-binding regions of MEILB2. To visualise this, and build the intervening 31 residues, we generated Alphafold2 models of the 2:2 complex between the full structured region of MEILB2 (amino-acids 22-338; herein referred to as MEILB2) and BRME1 . During modelling, we we conclude that the MEILB2-BRME1 has a linear structure in which the N-terminal BRME1-binding core is connected to C-terminal ARM domains by a MEILB2 2 coiled-coil ( Figure 4c).

MEILB2-BRME1 binds to DNA via its -cap
The surface electrostatics of the MEILB2-BRME1 2:2 model indicated that the N-terminal MEILB2-BRME1 core contains discrete patches of basic charge, whereas the C-terminal ARM domains are predominantly acidic (Figure 5a). Hence, we wondered whether the N-terminal end of the molecule may bind to DNA. Accordingly, electrophoretic mobility assays (EMSAs) showed that MEILB2-BRME1 forms discrete protein-DNA complexes with dsDNA and poly-dT ssDNA (Figure 5b,c). DNA-binding showed no overt sequence specificity, and saturation occurred at ratios of one MEILB2-BRME1 2:2 complex to 10 base pairs (dsDNA) or 20 nucleotides (ssDNA) (Figure 5b Notably, the basic charge of MEILB2-BRME1 is particularly concentrated at its N-terminal β-cap ( Figure 5a). Given that the β-cap stabilises the end of the coiled-coil, we hypothesised that it may serve as a rigid platform for DNA binding at the tip of the MEILB2-BRME1 complex. To test this, we introduced glutamate mutations of MEILB2 amino-acid K26, and BRME1 amino-acids R540 and R549, which are the main contributors of basic charge at the -cap. Consequently, DNA binding of MEILB2-BRME1 was abolished by both the MEILB2 K26E and BRME1 K540E R549E mutations (Figure 5f,g).
Thus, DNA-binding is mediated by the rigid -cap at the N-terminal tip of the MEILB2-BRME1 structure ( Figure 5h). Further, DNA-binding involves amino-acids from both MEILB2 and BRME1, so is a consequence of complex formation rather than being an intrinsic property of either component.

BRCA2-MEILB2-BRME1 acts as a DNA clamp
The physical separation between the two DNA-binding -caps of the BRCA2-MEILB2-BRME1 2:4:4 ternary complex suggested that may be able to bridge between DNA molecules. Accordingly, EMSAs demonstrated that BRCA2-MEILB2-BRME1 readily bound to a 75 base-pair dsDNA substate, forming protein-DNA networks that were too large to enter the gel (Figure 7a). To temper DNA-binding, and hence aid visualisation of protein-DNA species, we also analysed a BRCA2-MEILB2-BRME1 complex in which BRME1 was truncated to remove basic C-terminal amino-acids (deletion of 575-RKTK-579; herein referred to as BRME1 RKTK). This slightly diminished DNA-binding and reduced protein-DNA networks to sizes that were resolved by EMSA (Figure 7b).
We reasoned that if the DNA substrate were of sufficient length, it should be possible for the two caps of the ternary complex to bind cooperatively to the same DNA molecule. Hence, we analysed a 300 base-pair substrate, which has a length of 90 nm that far exceeds the 25 nm gap between caps. EMSAs showed that BRCA2-BRME1-MEILB2 initially formed discrete species with the 300 basepair substrate, and then generated protein-DNA networks when present at stoichiometric excess ( Figure 7c). This effect was particularly pronounced for the tempered BRCA2-BRME1-MEILB2 RKTK complex, which formed dominant discrete species prior to assembly at higher ratios into networks ( Figure 7d). These discrete intermediate species were not present for the 75 base-pair substrate ( Figure 7a,b), which has a length of 22 nm, so is insufficient to span between the two DNA-binding caps. These data strongly support a model in which the two -caps of the ternary complex act as spatially separated DNA-binding sites that can bridge between discrete DNA molecules.
The BRME1 R540 R549E mutation abrogated DNA-binding of the BRCA2-MEILB2-BRME1 complex (Figure 7e), confirming that DNA-binding of the ternary complex is mediated by the -caps. The MEILB2 K26E mutation also blocked binding of the BRCA2-MEILB2-BRME1 complex to the shorter 75 base-pair substrate (Figure 7f). However, it retained DNA-binding to the longer 300 base-pair substrate, albeit forming a single discrete species rather than protein-DNA networks (Figure 7g).
Hence, whilst the MEILB2 K26E mutation was sufficient to block DNA-binding by a single -cap, it likely retained sufficient residual affinity for the two -caps of the ternary complex to interact cooperatively with a sufficiently long single DNA molecule. Thus, the MEILB2 K26E mutant provides additional evidence in favour of our model.
In summary, BRCA2-binding induces dimerization of MEILB2-BRME1 into a V-shaped 2:4:4 assembly, in which DNA-binding -caps at the tips of its two limbs can independently interact with DNA molecules. Hence, BRCA2 induces MEILB2-BRME1 to act as a DNA clamp that holds together discrete DNA molecules, with a separation of approximately 25 nm (Figure 8). Thus, we propose that BRCA2 induces the clamping together of DNA molecules by MEILB2-BRME1 to facilitate inter-homologue recombination as part of the specialised mechanics of meiotic recombination.

Discussion
The molecular programme of meiosis utilises the machinery of DNA double-strand break repair by homologous recombination to align and generate crossovers between homologous chromosomes that are critical for their correct segregation and the formation of haploid germ cells. The particular adaptations of meiotic recombination require several additional meiosis-specific components. These include the MEILB2-BRME1 complex, which binds directly to BRCA2 and is essential for the correct localisation of RAD51 and DMC1 recombinases to meiotic DSBs. Here, we combine crystallographic and cell biology data to report the structure of the core MEILB2-BRME1 2:2 complex, which we combine with existing structural data to generate an experimentally validated model of BRCA2-MEILB2-BRME1 2:4:4 ternary complex. This V-shaped structure contains DNA-binding sites at the tips of its two limbs, which are separated by 25 nm, suggesting that it may function to clamp together DNA molecules. As BRCA2-binding is necessary for dimerization of MEILB2-BRME1 into the V-shaped structure, we conclude that its proposed role as a DNA clamp occurs within the context of its complex with BRCA2 ( Figure 8).
What is the function of the BRCA2-induced MEILB2-BRME1 DNA clamp in meiosis? We envisage two possibilities. Firstly, it may bridge between DSB ends, in a manner that favours their repair by interhomologue recombination. This is reminiscent of how MRN and CtIP are proposed to tether DNA ends and affect the choice of repair pathway at DSB sites (Oh and Symington, 2018, Davies et al., 2015, Wilkinson et al., 2019. Alternatively, it may bridge between homologous chromosomes to provide stabilising interactions between invading RAD51/DMC1-ssDNA nucleoprotein filaments and the template during inter-homologue recombination. This is an attractive model as it fulfils a unique requirement of meiosis, which is provided in mitotic recombination by cohesion between sister chromatids (Piazza et al., 2021). Notably, the 25 nm distance between DNA-binding sites of the dimerised MEILB2-BRME1 complex is comparable to the approximately 30 nm diameter of cohesin rings (Nasmyth, 2005). Hence, BRCA2-induced MEILB2-BRME1 DNA clamps may facilitate inter-homologue recombination by bridging between homologous chromosomes in the same manner as cohesin facilitates inter-sister recombination by tethering together sister chromatids.
A particular requirement of meiotic recombination is the removal of MEIOB2-SPATA22, in addition to RPA, from 5'-ssDNA overhangs for loading of RAD51 and DMC1 recombinases (Souquet et al., 2013, Ribeiro et al., 2021, Luo et al., 2013. As BRCA2-DSS1 can offload RPA (Bell et al., 2023), it was proposed that MEILB2-BRME1 may displace MEIOB2-SPATA22 during meiotic recombination . This is supported by the co-immunoprecipitation of MEIOB-SPATA22 with MEILB2, and the accumulation of MEIOB-SPATA22 at DSBs in MEILB2 and BRME1 knockouts (Zhang et al., 2019, Takemoto et al., 2020. Our finding that MEILB2-BRME1 binds directly to DNA, with no overt specificity for single-or double-stranded substrates, is consistent with this proposed role. Indeed, as BRCA2's MBD is proximal to its globular DNA-binding domain, we speculate that BRCA2-DSS1 and MEILB2-BRME1 may act together, in a coordinated manner, to offload RPA and MEIOB2-SPATA22 during meiotic recombination. This may occur alongside its proposed role in bridging between DNA ends or homologous chromosomes. Thus far, we have addressed how BRCA2-binding affects MEILB2-BRME1 structure, but we must also consider the converse issue of how MEILB2-BRME1 affects the structure of BRCA2. In isolation, BRCA2 can form monomers, dimers and larger oligomers, in a manner that involves its N-terminal and C-terminal regions, and is modulated by ssDNA, DSS1 and RAD51 (Le et al., 2020. Further, the BRCA2 dimer undergoes conformational change upon binding to RAD51 (Shahid et al., 2014). In meiosis, binding to MEILB2-BRME1 would clearly dimerise BRCA2 through its MBD region. However, given the lack of high-resolution structures of BRCA2 oligomers, it is not possible to say whether this would reinforce existing dimers or form alternative oligomers. In addition, binding to MEILB2-BRME1 may alter the conformation of BRCA2, such as to affect DMC1-binding and DNAbinding of the sites immediately downstream of the MBD. Any effects on the oligomeric state and conformation of BRCA2 likely favour inter-homologue rather than inter-sister recombination . Hence, these interactions are likely to be deleterious if they occur outside of meiosis.
Accordingly, MEILB2 is expressed in cancer cell lines and human tumour samples (Brandsma et al., 2019), and ectopic expression of MEILB2 inhibits homologous recombination in somatic cells (Sato et al., 2020. Whilst BRME1 is also upregulated in cancers and suppresses recombination upon ectopic expression , it is not yet possible speculate how it affects recombination as the structure and function of all but the MEILB2-binding site are unknown.
Overall, we propose that BRCA2-binding of MEILB2-BRME1 may clamp together resected ends or homologous chromosomes, may facilitate the removal of MEIOB-SPATA22 from 5'-ssDNA overhangs, and may alter the oligomeric state and/or conformation of BRCA2. These are not mutually exclusive, so may represent multifactorial roles of MEIB2-BRME1 in meiotic recombination. Further, MEILB2-BRME1 likely acts in coordination with the DMC1-binding site and globular DNA-binding domain that are immediately downstream of the MBD. Hence, the ternary complex formed by MEILB2-BRME1, BRCA2-DSS1, RAD51 and DMC1 may operate as a single functional unit that fulfils the above roles to facilitate inter-homologue recombination. Elucidating the structure of this meiotic 'recombinosome' will ultimately reveal the molecular mechanisms that underpin meiotic recombination. Further, the unusual architecture imposed by MEILB2-BRME1 may provide unique structural insights that will uncover the molecular basis of the wider role of BRCA2 in recombination-mediated DNA repair.

Size-exclusion chromatography multi-angle light scattering (SEC-MALS)
The absolute molar masses of protein samples were determined by multiangle light scattering coupled with size exclusion chromatography (SEC-MALS). Protein samples at >5 mg/ml were loaded onto a Superdex 200 Increase 10/300 GL size exclusion chromatography column (Cytiva) in 20 mM HEPES, pH 7.5, 150 mM KCl, 2 mM DTT for MEILB2-BRME1 constructs and in 20 mM HEPES, pH 7.5, 500 mM KCl, 2 mM DTT for BRCA2-MEILB2-BRME1 constructs, at 0.5 ml/min, in line with a DAWN HELEOS II MALS detector (Wyatt Technology) and an Optilab T-rEX differential refractometer (Wyatt Technology). Differential refractive index and light scattering data were collected and analyzed using ASTRA 6 software (Wyatt Technology). Molecular weights and estimated errors were calculated across eluted peaks by extrapolation from Zimm plots using a dn/dc value of 0.1850 ml/g. Bovine serum albumin (Thermo Fisher Scientific) was used as the calibration standard.

Size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS)
SEC-SAXS experiments were performed at beamline B21 of the Diamond Light Source synchrotron facility (Oxfordshire, UK). Protein samples at concentrations >8 mg/ml were loaded onto a Superdex™ 200 Increase 10/300 GL size exclusion chromatography column (GE Healthcare) in 20 mM HEPES pH 7.5, 150 mM KCl, 2 mM DTT for MEILB2-BRME1 constructs and in 20 mM HEPES pH 7.5, 500 mM KCl, 2 mM DTT for BRCA2-MEILB2-BRME1 constructs at 0.5 ml/min using an Agilent 1200 HPLC system. The column outlet was fed into the experimental cell, and SAXS data were recorded at 12.4 keV, detector distance 4.014 m, in 3.0 s frames. ScÅtter 3.0 was used to subtract, average the frames and carry out the Guinier analysis for the radius of gyration (Rg), and P(r) distributions were fitted using PRIMUS (P.V.Konarev, 2003). Crystal structures and models were fitted to experimental data using CRYSOL (Svergun D.I., 1995).

Electrophoretic mobility shift assays (EMSAs)
Protein complexes were incubated with 50 μM (per base pair) 75 bp and 300bp linear random sequence dsDNA, and 75 nt FAM-poly(dT) ssDNA, at concentrations indicated, in 20 mM HEPES pH 7.5, 325 mM KCl for 60 mins at room temperature. Glycerol was added at a final concentration of 3%, and samples were analysed by electrophoresis on a 0.4% (w/v) agarose gel in 0.5x TBE pH 8.0 at 20-40 V for ~4 h at 4 °C. DNA was detected by SYBR™ Gold (ThermoFisher).

K D determination by EMSA
Quantification of DNA-binding was performed though EMSA (as described above) using 50 nM FAMlabelled 75 bp random sequence dsDNA and 100 nM 75 nt poly(dT), at protein concentrations indicated. DNA was detected by FAM and SYBR™ Gold (ThermoFisher) staining using a ChemiDoc MP Imaging System (Bio-Rad). Gels were analysed using Image Lab software (Bio-Rad). The DNA-bound proportion was plotted against molecular protein concentration and fitted to the Hill-Langmuir equation (below), with apparent K D determined, using Prism8 (GraphPad). Protein concentrations used for K D estimation are quoted for the oligomeric species.

Structural modelling of the full MEILB2-BRME1 2:2 complex
Models were generated using a local installation of Alphafold2 v2.3.2  that was modified to control the use of PDB structures and newly solves crystal structures as templates.

Protein sequence and structure analysis
MEILB2 and BRME1 sequences were aligned and visualised using MUSCLE (Madeira et al., 2019) and Jalview (Waterhouse et al., 2009). Molecular structure images were generated using the PyMOL Molecular Graphics System, Version 2.4 Schrödinger, LLC.

Mice
Wild type mice were congenic with the C57BL/6J background. The mice are housed in IVC cages with a 12 hour dark and light cycle. The temperature is 20-22 °C and the relative humidity is between 45 and 60 %. The mice have bedding material in the form of wood shavings and wood litter as well as a house of paper and nesting pads as enrichment. Cage changing is done at least once a week. All animal experiments were approved by the Regional Ethics Committee of Gothenburg, governed by the Swedish Board of Agriculture (#1316/18).

Exogenous protein expression in the testis
Plasmid DNA was electroporated into live mouse testes as previously described (Shibuya et al., 2014). Briefly, male mice at postnatal day 16-20 were anesthetized with pentobarbital, and the testes were pulled from the abdominal cavity. Plasmid DNA (10 l of 5 g/l solution) was injected into each testis using glass capillaries under a stereomicroscope. Testes were held between a pair of tweezers-type electrodes (CUY21; BEX), and electric pulses were applied four times and again four times in the reverse direction at 35 V for 50 ms for each pulse. The testes were then returned to the abdominal cavity, and the abdominal wall and skin were closed with sutures. The testes were removed 24 h after the electroporation, and immunostaining was performed.

Immunostaining of spermatocytes
Testis cell suspensions were prepared by mincing the tissue with flathead forceps in PBS, washing several times in PBS, and resuspending in a hypotonic buffer (30 mM Tris (pH 7.5), 17 mM trisodium citrate, 5 mM EDTA, 2.5 mM DTT, 0.5 mM PMSF, and 50 mM sucrose). After 30 min, the sample was centrifuged and the supernatant was aspirated. The pellet was resuspended in 100 mM sucrose.
After 10 min, an equal volume of fixation buffer (1% paraformaldehyde and 0.1% Triton X-100) was added. Cells were applied to a glass slide, allowed to fix for 2 h at room temperature, and air-dried.
For immunostaining, the slides were incubated with primary antibodies in PBS containing 5% BSA for 2 h and then with the following secondary antibodies for 1h at room temperature: Donkey Anti-Rabbit Alexa 488 (1:1000; Invitrogen; A21206, 2376850) and Donkey Anti-Chicken Alexa 594 (1:1000; Invitrogen; A78951, 2551396). The slides were washed with PBS and mounted with VECTASHIELD medium with DAPI (Vector Laboratories).

Microscopy
Images were obtained on a microscope (Olympus IL-X71 Delta Vision; Applied Precision) equipped with 100× NA 1.40 objective, a camera (CoolSNAP HQ; Photometrics), and softWoRx 5.5.5 acquisition software (Delta Vision). Images were processed with Photoshop (Adobe).

Data availability
Crystallographic structure factors and atomic coordinates have been deposited in the Protein Data Bank (PDB) under accession number 7Z8Z, and raw diffraction data have been uploaded to https://proteindiffraction.org/.

Figure 1
Mechanics of homologous recombination.
Schematic of the key molecular players in mitotic (left) and meiotic (right) recombination. The main specialisations of meiotic recombination are DSB induction by SPO11-TOPOVIBL, coating of resected ends with MEIOB-SPATA22 (alongside RPA), the requirement for recombinase DMC1 (in addition to RAD51), and the role of MEILB2-BRME1 in BRCA2 function. These facilitate use of the synapsed homologous chromosome, rather than the cohesin-bound sister chromatid, as the primary repair template. In meiotic recombination, double Holliday junctions are resolved into crossovers and noncrossovers in a highly regulated manner. In mitotic recombination, crossovers are less frequent, and additional pathways operate that bypass the double Holliday junction intermediate.

Figure 2
Crystal structure of the MEILB2-BRME1 2:2 core complex.  The MEILB2-binding interface is required for BRME1 recruitment to meiotic DSBs.