Fig 2: (related to Fig 2). Independent binding of PRIMPOL, BTR, FANCM, and FANCD2 to ICL‐containing DNA AConfocal microscopy images of PrimPol and γH2AX immunofluorescence (IF) in control (UVA) or TMP‐UVA‐treated cells following downregulation of the indicated factors. Nuclear DNA is counterstained with DAPI. The UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average and SD of the percentage of cells that are PrimPol‐positive in the laser path in 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. The p‐values of the indicated comparisons are shown.BImmunoblots showing the levels of the indicated proteins after siRNA‐mediated downregulation. siBTR is a combination of siBLM, siTOP3A, siRMI1, and siRMI2. MEK2 is shown as loading control. Protein names in black and blue indicate the two different gels used.C–FConfocal microscopy images of BLM (C), RMI1 (D), FANCM (E), or FANCD2 (F) together with γH2AX IF staining in control (UVA) or TMP‐UVA‐laser irradiated WT or PRIMPOL KO cells, as in (A). Scale bar, 10 µm. Histograms (right panels) show the average percentage of cells positive for the recruitment of the indicated factor to the laser path. Average and SD of at least two assays are shown (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 the indicated comparisons are included. Source data are available online for this figure.

Fig 3: Identification of METTL16-regulated motifs in DNA-repair-related transcripts.a IGV snapshots of MeRIP-seq reads along indicated mRNAs. Red asterisk indicates differentially methylated sites identified by RADAR. b, c mRNA expression levels (b) and MeRIP-qPCR validation of m6A methylation levels (c) in the E13.5 in vitro cultured erythroblasts from control and Mettl16fl/flCreERT2+ (CKO) mice (n = 3 biological replicates). d Predicted secondary structures surrounding m6A peaks in Mat2a, Brca2, and Fancm mRNA. The ACAGAR boxes were shown in red and the m6A modification sites were shown in blue. e SELECT validation of the indicated m6A sites using total RNAs of the E13.5 cultured erythroblasts from control and Mettl16fl/flCreERT2+ mice (n = 3 biological replicates). f, g Dual-luciferase reporter assay to identify METTL16-regulated transcripts. f pmirGLO reporter vector encoding firefly and renilla luciferase were used. Indicated wild-type (WT) and mutant (Mut) sequences were inserted at the 3' end of the firefly luciferase. g Luciferase reporter activity of indicated reporters under METTL16 knockdown in HEK293T cells (n = 3 biological replicates). Renilla luciferase activity was used as an internal control. Firefly luciferase activity was further normalized to the value of the reporter harboring 3' UTR of Gapdh mRNA, which has been reported to have no m6A modification sites54. Data are expressed as mean ± SD (b, c, e, g). The p-values were calculated using two-tailed Student’s t-test (b, c, e, g). ns not significant. Source data are provided as a Source Data file.

Fig 4: ChIP-Seq analysis of genome-wide distribution of FANCM and GFP-DONSON.a Representative profile from chromosome 1, comparing RT (RT = log2(Early/Late)) in HeLa cells, A/B compartments as defined by the eigenvector calculated from Hi-C data from HeLa cells, and FANCM and GFP-DONSON distribution (enrichment = log2(ChIP/input)) in HeLa cells stably expressing GFP-DONSON. Some examples are shadowed in red of late-replicating regions aligning with FANCM enriched genomic regions. Some early-replicating regions are highlighted in blue showing correspondence with GFP-DONSON-enriched regions. In the RT profile, positive and negative values correspond to early and late replication, respectively. In the eigenvector profile, they correspond to the A and B compartments. Regions containing fragile sites are marked by red bars above the profiles. b Violin plot displaying the distribution of replication timing of 50-Kb genomic windows enriched in FANCM or GFP-DONSON ChIP (log2[ChIP/Input] > 0) and the A and B Hi-C compartments (eigenvector >0, <0, respectively), each compared with a matching number of randomly selected genomic windows of the same size. The box plot inside the violin plot shows the median (center line), the upper (Q3) and lower (Q1) quartiles (box bounds) and the highest and lowest values excluding outliers (extreme lines). c Coverage of H3K9me3, H3K4me3, FANCM, and GFP-DONSON of 50-Kb genomic windows within 25 replication-timing quantiles, going from late- to early-replicating regions, in HeLa cells expressing GFP-DONSON. d Coverage of H3K9me3, H3K4me3, FANCM, and GFP-DONSON of 50-kb genomic windows within 25 eigenvector quantiles, going from B to A Hi-C compartments in HeLa cells expressing GFP-DONSON. In the box plots in c, d, the center line represents the median, the box bounds represent the upper (Q3) and lower (Q1) quartiles, the extreme lines represent the highest and lowest value excluding outliers, and the black dot represents the mean.

Fig 5: Interactions of DONSON and FANCM with replisomes and chromatin in nontreated cells.a DONSON associates with some, but not all, replisomes in untreated cells. Chromatin was prepared from untreated GFP-DONSON-HeLa cells and sequential IP performed, first against GFP-DONSON, and then against the GINS protein PSF1 from the residual supernatant. Representative blot (n = 3). b PLA of GFP-DONSON and PSF1 demonstrates DONSON-associated replisomes are more frequent in early S phase than in late S phase in NT cells. Scored nuclei: PLA between GFP-D: PSF1 early S phase = 82, late S phase = 81, from three biological replicates. Data are mean ± s.e.m. c PLA between FANCM and MCM2 demonstrates low level of FANCM-associated replisomes in late S phase in nontreated cells. Scored nuclei: PLA between FANCM: MCM2 of early S phase = 73, late S phase = 75, from three biological replicates. Data are mean ± s.e.m. d IP of FANCM demonstrates low-level interaction with replisome protein MCM2. e PLA between GFP-DONSON and H3K4me3 or H3K9me3. Scored nuclei of GFP-DONSON and H3K4me3 in early S phase = 79, late S phase = 78. Scored nuclei of GFP-DONSON and H3K9me3 in early S phase = 77, late S phase = 82, from three biological replicates. Data are mean ± s.e.m. f PLA between FANCM and H3K4me3 or H3K9me3. Scored nuclei of FANCM and H3K4me3 in early S phase = 64, late S phase = 65. Scored nuclei of FANCM and H3K9me3 in early S phase = 68, late S phase = 65, from three biological replicates. Data are mean ± s.e.m. For PLA experiments in b, c, e, f, a two-sided Mann–Whitney rank-sum test was used to determine if differences were statistically significant. NS: not significant: P > 0.05. Source data are provided as a Source Data file.