Fig 1: RAD54L2 operates through a mechanism dependent on ZNF451 and independent of TDP2.(A) Biplot showing gene enrichment scores (NormZ) upon etoposide treatment in TP53KO (x axis) and TP53/RAD54L2KO (y axis) RPE-1 cells. Orange dots relate to well-characterized DDR factors, most discussed in the text. (B) Clonogenic survival assays of CTRL, RAD54L2KO, TDP2KO, and TDP2/RAD54L2KO RPE-1 cells upon treatment with etoposide. n = 3 independent experiments. Bars represent means ± SEM. Competitive growth assays of RPE-1 CTRL (C), LIG41KO (D), BRCA1KO (E), and ZNF451KO (G) cells transduced with virus expressing the indicated sgRNAs in the presence of various etoposide concentrations; n = 3 independent experiments. Bars represent means ± SEM. (F) Clonogenic survival assays of CTRL or ZNF451KO transduced with a CTRL sgRNA or with an sgRNA targeting RAD54L2. n = 3 independent experiments. Bars represent means ± SEM. (H) Extracts of etoposide-treated CTRL or ZNF451KO RPE-1 cells stably expressing mCherry [empty vector (EV)] or mCherry-RAD54L2WT were subjected to mCherry IP followed by Western blotting for the indicated proteins. This experiment was repeated three times with similar results.
Fig 2: RAD54L2 binds SUMO and TOP2α/β and requires its SIMs to interact with SUMO-modified TOP2α/β.(A and B) Volcano plots showing RAD54L2 interactors identified by IP-MS in untreated (UT) cells (A) or etoposide (ETP)–treated cells. (B) Green dots bordered by dotted lines indicating P values ≤0.05 and log2 fold changes ≥2 are significant interactors. (C) Schematic of the putative domains and relevant properties of RAD54L2. The K311A catalytic site mutation is labeled, and the SIMs are highlighted in blue dots. (D) Clonogenic survival assays of control (CTRL) cells or RAD54L2KO cells complemented with vectors expressing mCherry, mCherry-RAD54L2WT, mCherry-RAD54L2K311A mutant, or mCherry-RAD54L2ΔSIM mutant; n = 3 independent experiments. Bars represent means ± SEM. (E) Coimmunoprecipitation (IP) of mCherry from extracts of untreated or etoposide treated RPE-1 cells stably expressing mCherry (EV), mCherry-RAD54L2WT, or mCherry-RAD54L2ΔSIM mutant. This experiment was repeated six times with similar results.
Fig 3: RAD54L2 counters TOP2ccs by promoting their resolution.(A) Quantification of γH2AX foci in G1 cells (cyclin A negative). Cells were treated with 30 μM etoposide for 30 min, washed, and left to recover for 4 or 8 hours. n > 3 independent experiments. Bars represent means ± SEM. One-way analysis of variance (ANOVA) was used for statistical testing. (B) DUST assays in CTRL or RAD54L2KO HAP1 cells collected after 1 hour of treatment with 20 μM etoposide or 30 min or 1 hour after etoposide washout. This experiment was carried out three times with similar results. (C) Quantifications of TOP2α or TOP2β signal from the DUST assays. Bars represent means ± SEM. (D) Proposed mechanism for RAD54L2 in TOP2cc resolution. UBC9, SUMO conjugating enzyme 2 ligase. Figure generated with BioRender. ns, not significant.
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