High Resolution View on the Regulation of Recombinase Accumulation in Mammalian Meiosis

Abstract

A distinguishing feature of meiotic DNA double-strand breaks (DSBs), compared to DSBs in somatic cells, is the fact that they are induced in a programmed and specifically orchestrated manner, which includes chromatin remodeling prior to DSB induction. In addition, the meiotic homologous recombination (HR) repair process that follows, is different from HR repair of accidental DSBs in somatic cells. For instance, meiotic HR involves preferred use of the homolog instead of the sister chromatid as a repair template and subsequent formation of crossovers and non-crossovers in a tightly regulated manner. An important outcome of this distinct repair pathway is the pairing of homologous chromosomes. Central to the initial steps in homology recognition during meiotic HR is the cooperation between the strand exchange proteins (recombinases) RAD51 and its meiosis-specific paralog DMC1. Despite our understanding of their enzymatic activity, details on the regulation of their assembly and subsequent molecular organization at meiotic DSBs in mammals have remained largely enigmatic. In this review, we summarize recent mouse data on recombinase regulation via meiosis-specific factors. Also, we reflect on bulk "omics" studies of initial meiotic DSB processing, compare these with studies using super-resolution microscopy in single cells, at single DSB sites, and explore the implications of these findings for our understanding of the molecular mechanisms underlying meiotic HR regulation.

Keywords: ChIP-seq; DSB repair; meiosis; recombinase; super-resolution microscopy.

FIGURE 1

Most commonly used fluorescent super-resolution microscopy techniques in meiosis research. (A) Structured illumination microscopy (SIM) – SIM uses a movable diffraction grating in combination with widefield microscopy. By exciting the sample and simultaneously translating and rotating the grid, an interference pattern—also called a Moiré pattern—occurs which contains more detailed information. Using mathematical tools these images are converted to one image with a lateral resolution up to ∼115 nm (Guerra, 1995; Gustafsson, 2000). (B) Stimulated emission depletion (STED) – In addition to the excitation laser, STED microscopy includes a second laser that generates a donut shaped STED beam of a longer wavelength that induces stimulated emission of any fluorophore that is not located at the center of the STED beam. This physically narrows the point spread function and therefore increases the lateral resolution up to ∼70 nm (Galbraith and Galbraith, 2011). (C) Direct Stochastic optical reconstruction microscopy(dSTORM) – dSTORM microscopy can provide high resolution images with a lateral resolution of ∼20 nm. Fluorophores are stochastically activated by increasing the switch between active and dark-state using an oxygen-reducing buffer and high laser power. Images are generated by combining individual localization signals which are recorded over time (Rust et al., 2006). It should be noted that although single molecules can be detected, the exact number of target proteins present cannot be quantified due to the indirect detection method, using fluorescent antibodies. (D) Expansion microscopy (ExM) – Expansion microscopy is a method to increase resolution by enlargement of the sample. This enlargement makes use of a swelling polymer that expand (swelling polymer) samples directly or indirectly (Chen et al., 2015). ExM can be combined with other microscopy techniques resulting in ExM-SIM, ExSTORM or ExSTED with a resolution increase of ∼3–4x fold (Gao et al., 2018; Wang et al., 2018; Xu et al., 2019; Zwettler et al., 2020).

FIGURE 2

Schematic models interpreting bulk and single-cell data on recombinase loading at individual DSBs in mouse meiosis. (A) (left)The location of initial RAD51 and DMC1 loading on ssDNA might be random. The suggested directionality to their filament formation (Brown and Bishop, 2014) could subsequently lead to the RAD51 filaments extending in 3′–5′ direction while DMC1 filaments extending in 5′–3′ direction. The resulting ssDNA would then be occupied with a varying composition of RAD51 and DMC1 recombinases as represented in 1–3. It is equally possible that multiple short stretches of RAD51 and DMC1 filaments are assembled on ssDNA as shown in 4. (right) is a schematic drawing of putative RAD51 and DMC1 ChIP-seq reads corresponding to their loading on individual break sites as depicted in 1–4. The bulk cell nature of ChIP-seq analysis would then yield density plots as indicated in the cartoon, inspired from the RAD51 and DMC1 ChIP-seq analysis from Hinch et al. (2020). (B) Super-resolution imaging of DSB repair foci in mice revealed various configurations of DMC1 (D, red oval) and RAD51 (R, green oval). The most frequent configuration, D1R1, can be interpreted in three ways: Assuming that the recombinase foci with a preferred nearest neighbor distance of around ∼800 nm (Slotman et al., 2020) represent two ends of DSB, D1R1 can be interpreted as DMC1-RAD51 pairs loaded symmetrically across two ends of DSB. Alternatively, D1R1 configuration might also be the result of two ends of DSB each loaded with DMC1-RAD51 pairs that end up so close (<50 nm) that they could not be resolved even with super-resolution microscopy. It is equally possible that one end of DSB might be occupied by other ssDNA binding proteins or is being processed while the other end is loaded with DMC1-RAD51 pair. Secondly, based on the paired foci observed in other species and according to ChIP-seq data, a D2R2 can be interpreted as a subset of D1R1 loaded ends of a DSB that lie within the ROI (<600 nm). However, this configuration is rare. Besides these configurations, other configurations such as D2R1 and D1R2 (of which D2R1 is the second most frequent configuration) consisting of a smaller DMC1 or RAD51 foci along with a D1R1 have been observed rather frequently. These configurations may be interpreted as the presence of a single DMC1 or RAD51 focus on the other end of DSB occupied by a D1R1, or an extra focus on the same ssDNA accommodating a D1R1. Other interpretations of these recombinase configurations are also possible. ROI: region of interest Scale bar representing 250 nm. Binary super-resolution images of RAD51 and DMC1 configurations are derived from Slotman et al. (2020).