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. 2022 Sep 15;50(17):9909–9929. doi: 10.1093/nar/gkac746

Figure 6.

Figure 6.

RAD51-dependent H-shaped structures accumulate in genomic DNA 2 h following UVC challenge. (A) Examples of in vivo five-branched structures (H-shaped) observed by TEM 2 h after UVC (25 J m–2) in XPV cells. Inlays represent enlargements of the regions indicated by dotted arrows. The central bar of the H-shaped structures can vary in length and be made of dsDNA (i, ii, vii), ssDNA (iii, viii) or both (iv). Very rarely, open-centred structures reminding Holliday Junctions were observed (v, vi). These structures can be located next to the elongation point of a fork (vii, viii, ix). (B) Relative levels of H-shaped structures (normalized to the count of 100 RFs). Values are the mean ± SD of two independent experiments (ordinary one-way ANOVA followed by Tukey's multiple comparisons test, **P < 0.01, ***P < 0.001). TEM analysis was performed in XPV cells 2 h after UVC (25 J m–2). When indicated, cells were pre-treated with 20 μM B02 for 8 h. (C) Lengths of the H-shaped structure central bars (pool from the two independent experiments, bar is the median of the distribution, Kruskal-Wallis with Dunn's multiple comparisons test, ns: not significant). (D) Examples of joint molecules formed in the in vitro RAD51-dependent strand-exchange assay observed by TEM. The circular ssDNA Phix174 virion was incubated with RAD51, followed by RPA addition, then linearized Phix174 RFI homologous plasmid was added to the reaction. RAD51 polymerizes onto the ssDNA substrate to form the presynaptic filament and then catalyses the strand invasion and strand transfer with the linear homologous dsDNA. Final products are linear ssDNA (lss) and nicked dsDNA plasmid (nc). For TEM observations, reactions were subjected to UVA/trioxalen in vitro crosslinking, deproteinization, and spread using the BAC hyperphase method. (i) circular ssDNA Phix174 virion (css) and linearized homolog plasmid (lds). (ii), (iii) Representative views of joint molecules. Formation of five-branched (H-shaped) structures is shown in (iii). (iv) Final products of the reaction. Scale bar = 200 nm.