Characterization of RAD51AP1 isoforms. (A) The three human RAD51AP1 isoforms used in this study. (B) SDS-PAGE analysis of the purified tagged RAD51AP1 isoforms. (C) In a pull-down assay, GST-tagged RAD51AP1 Isoform 2 was found to associate with human DMC1 (lane 3) but not with S. pombe Dmc1 (SpDmc1; lane 6). Control reactions were carried out with GST. The supernatant (S), wash (W), and SDS eluate (E) of the pull-down reactions were analyzed by SDS-PAGE. (D) RAD51AP1 isoform 2 specifically stimulates D-loop formation by human DMC1. (I) Schematic of the D-loop assay. (II) RAD51AP1 (50, 100, 200, 300, or 500 nM) enhanced the D-loop reaction mediated by human DMC1 (lanes 3 to 7) in an ATP-dependent fashion (lane 8). SpDmc1 was refractory to RAD51AP1 (200, 300, or 500 nM; lanes 9 to 11) but was enhanced by ScRad54 (200 nM; lane 12) in an ATP-dependent fashion (lane 13). (III) The results were plotted; error bars represent the standard deviation (± SD) calculated based on at least three independent experiments.

Lack of physical or functional interaction of RAD51AP1 Isoform 3 with DMC1. (A) Affinity pull-down analyses using GST-tagged RAD51AP1 (I) and MBP-tagged RAD51AP1 (II) isoforms revealed that isoform 3 does not interact with DMC1 (I, lane 9). Control pull-down reactions using GST and MBP were also included. The supernatant (S), wash (W), and SDS eluate (E) fractions of the pull-down reactions were analyzed by SDS-PAGE. (B) Although RAD51AP1 isoform 1 (lanes 8 and 9) and isoform 2 (lanes 4 and 5) were equally capable of enhancing the DMC1-mediated D-loop reaction in an ATP-dependent manner (lanes 6 and 10), RAD51AP1 isoform 3 (lanes 12 and 13) was devoid of such ability. The amount of each RAD51AP1 isoform used was 400 or 600 nM. The percentages of D-loop products formed were quantified and plotted. (C) Isoform 1 and isoform 2 were both found capable of functional synergy with DMC1 in duplex-DNA capture (lanes 4 and 7) in an ATP-dependent fashion (lanes 5 and 8); isoform 3 was much less effective (lane 10). Various controls were included. The amount of each RAD51AP1 isoform used was 600 nM. The results were plotted. (D) Although RAD51AP1 isoform 1 and isoform 2 were both capable of functional synergy with DMC1 in synaptic complex assembly (lanes 5, 6, 9, and 10) in an ATP-dependent fashion (lanes 7 and 11), isoform 3 was much less effective (lanes 13 and 14). Various controls were included. The amount of MBP-tagged RAD51AP1 isoform 1 and GST-tagged RAD51AP1 isoform 2 or isoform 3 used was 300 or 500 nM. The results were plotted. (B–D) Error bars represent the standard deviation (± SD) calculated based on at least three independent experiments.

Functional synergy of RAD51AP1 and DMC1 in duplex capture and synaptic complex assembly. (A) Schematic of the duplex capture reaction. (B) DMC1 alone (lane 2) or RAD51AP1 alone (lanes 3 and 4) exhibited little duplex capture activity. DMC1 synergized with RAD51AP1 (300 or 600 nM) in duplex-DNA capture (lanes 5 and 6), with a dependence on ATP (lane 7). The percentages of captured DNA were quantified and plotted in (II). Error bars represent ± SD calculated based on at least three independent experiments. (C) The basis for the protection of the target DNA against digestion by the restriction enzyme SspI is illustrated. (D) RAD51AP1 (0.5 or 1 μM) was incubated with the DMC1 presynaptic filament assembled on either the homologous ssDNA (I) or heterologous ssDNA (II), and the protection of the dsDNA against digest by Ssp1 was assessed (lanes 6 and 7). Various controls (lanes 2–5) were included. DNA protection was contingent upon ATP being present (lane 8). The results were plotted. Error bars represent ± SD calculated based on at least three independent experiments.

RAD51AP1 colocalizes with DMC1 foci on meiotic chromatin. (A) Example of a spermatocyte nucleus in leptonema. Chromosome spreads were stained with anti-SYCP3 (blue), anti-DMC1 (red), and anti-RAD51AP1 GP49 (green) antibodies. Scale bar = 10 μm. (B–D) Zoomed-in displays of regions indicated in A that harbored colocalized foci of RAD51AP1 and DMC1. Note that foci colocalization was scored without restriction to the subset of foci that share the same centroid. (E and F) Rotation experiment evaluating the statistical significance of RAD51AP1-DMC1 colocalization. E is a zoomed-in display of the 20 × 20 μm box marked in A within which colocalized RAD51AP1 and DMC1 foci were counted, as indicated by white arrows. F corresponds to the same region in E but with the RAD51AP1 immunofluorescence channel rotated 180°. RAD51AP1 foci that overlap with DMC1 foci after image rotation, as indicated by white arrowheads, are considered fortuitously colocalized. (G) Counts of axis-associated DMC1, RAD51AP1, and colocalized foci in leptotene stage spermatocytes. Each dot represents the count from one nucleus and the same 16 nuclei were quantified (bars = mean ± SD). (H) Colocalized foci as a percentage of DMC1 or RAD51AP1 total foci. Each dot represents the percentage from one nucleus and 16 nuclei were quantified (bars = mean ± SD). (I) Results of a rotation experiment wherein axis-associated DMC1 or RAD51AP1 foci were counted before and after 180° rotation of the DMC1 or RAD51AP1 immunofluorescence channel. Each dot represents the count from one nucleus and eight nuclei were quantified (bars = mean ± SD). (J) Colocalized foci as a percentage of DMC1 total foci before and after 180° rotation of the RAD51AP1 immunofluorescence channel. Each dot represents the count from one nucleus and eight nuclei were quantified (bars = mean ± SD).