The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates

Abstract

Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.

Keywords: D-loop; DMC1; DNA double-strand breaks; DNA repair; RAD51; RPA; crossover; meiosis; recombination; strand invasion.

Graphical abstract

Figure 1

Genome-wide Maps of RAD51, RPA, and DMC1 Binding in Meiosis (A) PRDM9 binds DNA at particular sequence motifs, some of which become sites of breaks by SPO11. 5′-to-3′ strand resection occurs, creating ssDNA overhangs, which are bound first by RPA (blue) and then by RAD51 and DMC1 (shown in gray to indicate that their relative localization is unknown). Our ChIP-seq maps measure complexes of these proteins with ssDNA (but not dsDNA). (B) An Integrative Genomics Viewer illustration of SPO11 oligos (measuring DSBs, black), DMC1 (red), RAD51 (green), and RPA (blue) ssDNA ChIP-seq reads in B6 in a segment of chromosome 1 with two hotspots ~18 kb apart. (C and D) Comparison of measures of RAD51 and RPA binding (C and D, respectively), with the number of SPO11 oligos in autosomal hotspots identified using DMC1 (n = 16,926) in B6. The intensity of RAD51 and RPA binding is a strand-aware and background-adjusted measurement of the number of ChIP-seq reads. (E and F) Comparison of measures of RAD51 and RPA binding (E and F, respectively), with DMC1 binding (Hinch et al., 2019) in autosomal hotspots (n = 23,631) in the hybrid.

Figure 2

RAD51 and DMC1 Bind ssDNA on the DSB-Initiating Chromosome with Distinct Localization, whereas RPA Binds the DSB-Initiating and Repair Template Chromosomes (A) Heatmaps showing coverage of SPO11 oligos (black), DMC1 (red), RAD51 (green), and RPA (blue) ChIP-seq reads in hotspots identified by DMC1 ChIP-seq in B6. Each row represents one of the 4,000 most active hotspots with a well-defined PRDM9-binding motif, ordered by the number of SPO11 oligos. The coverage shown is centered relative to the midpoint of each PRDM9-binding site. The numerical coverage values are dependent on experimental factors (e.g., sequencing depth) and are not comparable between experiments. (B) Density of DMC1 binding on the DSB-initiating (light red) and repair template (dark red) homologs relative to DSB sites, separated for Watson (left) and Crick (right) strands. Density was inferred in asymmetric PRDM9^CAST hotspots with a well-defined PRDM9-binding motif in the hybrid (n = 1,955) via deconvolution and normalized so that the area under the curves for each strand is 1 (100-bp smoothing). (C) As in (B) but for RAD51 binding on the DSB-initiating (light green) and repair template (dark green) homologs, separated for Watson (left) and Crick (right) strands. (D) As in (B) but for RPA binding on the DSB-initiating (light blue) and repair template (dark blue) homologs, separated for Watson (left) and Crick (right) strands.

Figure 3

RAD51 and DMC1 Foci Form Paired Co-foci (A–C) Representative images from structured illumination microscopy of spread spermatocytes in different meiotic stages, stained for DMC1 (red), RAD51 (green), and SYCP3 (white) in 13–15 dpp (days postpartum) B6 × DBA/2J mice. SYCP3 is a major component of chromosome axes (Baudat et al., 2013). Scale bars, 5 μm (500 nm for magnified images).

Figure 4

Relative Binding of DMC1, RAD51, and RPA on the DSB-Initiating Homolog (A) Comparison of observed RPA binding on the DSB-initiating homolog (blue) with the best-fitting linear combination of DMC1 and RAD51 binding inferred by modeling (65% and 35%, respectively; the Crick strand is shown; see also Figure S6A). (B) Composition of the ssDNA nucleoprotein filament inferred by modeling (Crick strand). Shown is RPA on the DSB-initiating homolog (light blue background) with inferred proportions of DMC1 (red) and RAD51 (green). RPA is normalized so that the area under its curve is 1, with DMC1 and RAD51 proportions being 0.65 and 0.35, respectively (100-bp smoothing). (C) Comparison of measures of RPA (left), DMC1 (center), and RAD51 (right) binding, with the SPO11-oligo count in autosomal (n = 16,926) and non-pseudoautosomal X chromosome hotspots (n = 1,103, orange) in B6. (D) Density of RPA (left), DMC1 (center), and RAD51 (right) in hotspots where DMC1 binding is greater on one strand (orange) than on the other strand (purple) in B6. Only skewed hotspots with a well-defined PRDM9 motif and over 8 kb away from the nearest hotspot were included (n = 6,340). For purposes of illustration, all skewed hotspots were co-oriented to have greater binding on the left. These plots show protein binding relative to break sites (after deconvolution) and are thus not confounded by DSB localization (see also Figure S7B).

Figure 5

RPA Binds the Repair Template Chromosome within Inter-homolog Recombination Intermediates, which May Be Resolved as Crossovers or Non-crossovers (A) Comparison of RPA (blue) on the repair template homolog, with DMC1 (red) and RAD51 (green) on the DSB-initiating homolog, relative to the break site (solid black tick). Dotted lines indicate positions of peak DMC1 (red) and RAD51 (green) binding (only the Crick strand is shown for clarity). (B) Model of RPA binding on the repair template chromosome. DMC1 (red) and RAD51 (green) bind ssDNA overhangs on the DSB-initiating chromosome (black), with DMC1 close to the DSB and RAD51 nearer the junction with dsDNA. A segment of the DMC1-bound nucleoprotein filament invades and pairs with the repair template chromosome (light blue). The corresponding strand of the repair template chromosome becomes single stranded (known as the displacement loop or D-loop, highlighted in yellow) and is bound by RPA (dark blue). The D-loop is extended, potentially after further unwinding of the DNA duplex and binding of RPA ahead of the DNA polymerase. (C) Comparison of crossover resolution probability and RPA signal on the repair template chromosome between hotspots in different regions of chromosomes (for the same estimated number of DSBs). Asymmetric hotspots in the hybrid (n = 4,218) were divided into 4 bins with an equal RPA signal on the DSB-initiating chromosome, based on distance to the centromere. The proportion of crossovers (orange) and RPA on the repair template (blue) is shown for each bin. The proportion expected from the number of DSBs in each bin is 0.25 (dotted black line). Error bars show 95% confidence intervals estimated using 100,000 bootstrap iterations. The number of asterisks indicates the significance (n asterisks indicate p < 10⁻ⁿ). The numbers below the bars indicate the estimated fraction of inter-homolog recombination intermediates that are repaired as crossovers (STAR Methods). (D) Comparison of RPA binding on the repair template chromosome on the same side as the invading strand (dark blue; i.e., to the right of the break site on the Crick strand and left of it on the Watson strand), RPA on the other side of the break site (light blue), with the probability of a site being within crossover-associated (orange) and non-crossover-associated (gray) gene conversion tracts. Curves were normalized to have area 1 under each curve to facilitate comparison.