Figure 4.

RAD51 binds to the RAD51AP1/NCP complex, RAD51AP1 stimulates duplex capture with the NCP and is recruited to the chromatin fraction in human cells after DNA damage. A, schematic of the duplex capture assay with His₆/FLAG-tagged RAD51AP1 and either nucleosome-free DNA (i.e., 147 bp dsDNA) or the NCP. B, qualitative analysis of captured DNA (beads) and DNA in supernatant by agarose gel electrophoresis. C, quantitative analysis: Symbols are the results from independent experiments. Bars are the means from three independent experiments ±1 SD; ∗, p < 0.05; ∗∗, p < 0.01; two-way ANOVA. D, schematic of the D-loop reaction with chromatinized pBluescript II SK (-) plasmid DNA. E, addition of RAD51AP1 (50, 100 and 200 nM; lanes 2–4) or RAD54 (100 and 200 nM; lanes 5–6) promotes the RAD51-mediated D-loop reaction on chromatinized DNA. F, quantification of the results. Symbols are the results from independent experiments. Bars are the means from two to four independent experiments ± 1SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; multiple t test analysis. G, agarose gel to show that wild-type RAD51AP1 (100 and 200 nM; lanes 2–3) promotes the RAD51-mediated D-loop reaction on chromatinized DNA, but RAD51AP1-K3WA (100 and 200 nM; lanes 4–5) and RAD51AP1-K7WA (100 and 200 nM; lanes 6–7) are unable to do so. H, western blots of fractionated extracts of the nuclei from HT1080 and U2OS cells without and after exposure to mitomycin C (MMC). The signals for ß-Actin and histone H3 serve as loading and fractionation control, respectively. RAD54 is shown for comparison purposes.