Fig 2: PrimPol is rapidly recruited to ICLs in an RPA-dependent manner Immunoblots show the levels of the indicated proteins in whole cell extracts (WCE), soluble and chromatin-enriched fractions. Tubulin and CTCF are shown as controls for soluble and chromatin-bound proteins, respectively. The position of the ubiquitylated form of FANCD2 (Ub-FANCD2) is indicated. Ponceau S staining is shown as a loading control.Confocal microscopy images of PrimPol and ?H2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-laser irradiated (10 µM TMP, 2 h followed by UVA laser irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of three assays (n = 100 cells per TMP-treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. P-values of individual comparisons are indicated.Confocal microscopy images of PrimPol and ?H2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated cells following control or RPA2 downregulation. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average PrimPol-positive cells and SD of three assays (n = 100 cells per TMP-treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. P-values of individual comparisons are indicated.Confocal microscopy images of PrimPol and ?H2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated PRIMPOL KO cells stably expressing V5-tagged WT or RPA binding domain mutant (RBDm) PrimPol versions. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of two assays (n = 100 cells per condition in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. P-values of individual comparisons are indicated.Confocal microscopy images of PrimPol and ?H2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated WT and BLM KO cells upon downregulation of TOP3A, RMI1, and RMI2 (siTR) when indicated. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of three assays (n = 250 cells per condition). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test.Immunoblots show the levels of BLM, TOP3A, RMI1, RMI2, and PrimPol after TOP3A, RMI1, and RMI2 downregulation in WT and BLM KO cells. Ponceau S is shown as loading control. Source data are available online for this figure.

Fig 3: Mutations in TOP3A Markedly Reduce Amounts of TopIIIa but Do Not Affect Its Intrinsic Decatenation Activity(A) Schematic of TopIIIa with locations of variants annotated. Colors are as follows: red, TOPRIM domain; purple, TOPA domain; and blue, GRF zinc-finger domain.(B) TOP3A mutations markedly reduce protein amounts. An immunoblot of a polyacrylamide gel is shown and was generated from total cell lysates from dermal fibroblast lines derived from affected individuals probed with an anti-TopIIIa antibody. siRNA depletion of TOP3A in a fibroblast line from control 2 was used to confirm the specificity of the antibody. C1 and C2 are unrelated control fibroblast lines. TopIIIa antibody was raised against amino acids 652–1,001.(C) Recombinant TopIIIaThr812LeufsTer101 (denoted TopIIIaP1) is less stable than TopIIIaWT. An immunoblot of a polyacrylamide gel is shown and was generated from purified TopIIIaWT and TopIIIaP1 proteins probed with an anti-TopIIIa antibody. Protein size markers are 250, 170, 140, 100, and 70 kDa.(D) Schematic depicting the BTRR complex and its role in dHJ dissolution. Homologous sister chromatids are shown in red and blue.(E and F) TopIIIaP1 is proficient for promoting dHJ dissolution with BLM in combination with RMI1 and RMI2. (E) Representative polyacrylamide gel demonstrating the enzyme-concentration dependence of the dHJ dissolution activity of TopIIIaWT and mutant (TopIIIaP1) TRR complexes with 20 nM BLM at 37°C in 30 min. TRR was included in reactions at increasing concentrations ranging from 20 pM to 10 nM. A labeled circular oligonucleotide (lane 1) and dHJ (lane 2) were included as markers. (F) Quantification of the dHJ dissolution reaction shown in (E). The reaction was repeated twice more with very similar results.

Fig 4: Topoisomerase 2a and 2ß co-localize with replicating mitochondrial DNA nucleoids. (A) Detection of topoisomerases in mitochondrial extracts of human and mouse cell lines by Western blot. Human cell lines: HEK293T and HeLa. Mouse cells: proliferating C2C12 myoblasts and differentiated C2C12 myotubes, mouse embryonic fibroblasts of wildtype and Top2ß (–/–) knockout mice. ATAD3 is used as loading reference. Note that the used polyclonal antibodies might detect mouse and human proteins with different efficiencies. (B) Detection of topoisomerase Top2ß, Top1mt and Top3a in mitochondrial extracts of various mouse tissues by Western blot. TFAM is used as indicator of mtDNA content, while TOMM20 serves as mitochondrial loading control. (C) Localization of mitochondrial topoisomerases in floatation gradients of mitochondrial extracts from HEK293T cells. The pellet fraction after digitonin treatment contains inner membrane-associated proteins and nucleoids, while the supernatant fraction contains soluble nucleoids and proteins without membrane-association. Top2a and Top2b co-localize well with the membrane-attached, replicating mtDNA nucleoids characterized by the presence of the mitochondrial helicase TWNK. Also the mitochondrial topoisomerases Top3a and Top1mt partially co-localize with this fraction, but a larger proportion is found in other fractions. (D) Mitochondrially targeted Top2ß co-localizes with replicating nucleoids. HeLa cells transiently transfected with an expression vector for myc-tagged, mitochondrially targeted Top2ß were immuno-stained with a monoclonal antibody against the myc-tag (green) and a polyclonal antibody against mtSSB (red) as a marker for mtDNA replication.

Fig 5: FANCD2-dependent BLM regulation is conserved in human cells. (A) Human BLM forms a constitutive complex with non-ubiquitinated FANCD2. Left panel: WCE were prepared from PD20 + D2 cells (lanes 1 and 2) and subjected to IP with rabbit IgG (lane 3), FANCD2 antibody (lanes 4 and 5) or BLM antibody (lanes 6 and 7). Input and equal volumes of IP samples were analyzed for presence of FANCD2 and BLM. Right panel: The same samples shown in the left panel were reanalyzed by extended gel electrophoresis. Sample volumes were adjusted to contain equal amounts of FANCD2 to better discern between non-ubiquitinated and monoubiquitinated FANCD2 isoforms. N.B.: to achieve equal FANCD2 protein concentrations in WCE and IP lanes, 1/30 of the original sample volumes were loaded in lanes 1, 2, 4 and 5. (B) Left panel: Human FANCD2 supports BLM protein stability. WCE were prepared from human PD20 + D2 cells (lanes 1 and 2) and PD20 cells (lanes 3 and 4) that had been untreated (lanes 1 and 3) or treated with 30 µM APH (lanes 2 and 4) and analyzed for FANCD2 and BLMcx members. Middle panel: Human FANCD2 mediates BLMcx chromatin recruitment. PD20 + D2 cells (lanes 1 and 3) and PD20 cells (lanes 2 and 4) were untreated (lanes 1 and 2) or treated with 30 µM APH for 3 h (lanes 3 and 4). Chromatin fractions were isolated from the cells and analyzed for presence of FANCD2, BLM and TOP3a. Histone H3: loading control. Right panel: Human FANCD2 is crucial for BLMcx assembly. WCE from untreated or APH-treated PD20 + D2 and PD20 cells were subjected to IP with rabbit IgG (lanes 1 and 4) or BLM antibody (lanes 2, 3, 5 and 6), and IP samples were analyzed for the presence of FANCD2 and BLMcx members.