Fig. 5: TRESLIN-MTBP is a limiting firing factor that defines IZs.

a Immunoblot showing the protein levels of replication factors. The endogenous genes encoding the indicated proteins were tagged with a 3×FLAG epitope. The FLAG-tagged proteins are marked with an open arrowhead. Antibodies against the FLAG epitope and tubulin were used for detection. Two biological replicates were performed, and a representative result is shown. b Representative profiles of LD-OK-seq (S1), MCM4, and TRESLIN ChIP-seq, RNA-seq, and H3K9me3. Okazaki fragment reads from the Crick and Watson strands are depicted in red and blue, respectively. Early-firing IZs are highlighted in pink. All ChIP-seq data are presented as input-normalised signal ratios. Cells for TRESLIN ChIP-seq were collected 6 h after release from nocodazole. TRESLIN ChIP-seq profiles are shown for control conditions and GINS4-depleted conditions (-GINS4). RNA-seq data are displayed as log₂-transformed TPM values. Genes are annotated with coloured arrows indicating transcriptional directions. c Top: Schematic illustrating an intergenic region with or without IZ within Early- or EtoM-RT domains. Both regions are flanked by actively transcribed genes and loaded with MCM-DH, but only the intergenic region with IZ fires in early S phase. Bottom: LD-OK-seq RFD (S1) at intergenic regions categorised by the presence or absence of IZs. Intergenic regions are centered with 100 kb extensions on both sides and ordered by size. d Heatmaps of ORC1, MCM4, and TRESLIN ChIP-seq signals across intergenic regions categorised by the presence or absence of IZs. e Representative LD-OK-seq profiles in WT (S1–6) and TRESLIN-MTBP overexpressing cells (T/M-OE, S1-S2). Early-firing IZs and mid-firing IZs in WT cells are shaded in pink and green, respectively. Arrowheads link IZs to corresponding positions in the T/M-OE LD-OK-seq tracks. f Representative LD-OK-seq (S1) and TRESLIN ChIP-seq (-GINS4) profiles in WT and RIF1-KO cells. IZs in WT cells are marked with green boxes. ChIP-seq data are presented as input-normalised signal ratios. g Heatmaps showing TRESLIN enrichment (-GINS4) around IZs in WT and RIF1-KO cells. IZs are centered with 100 kb extensions on both sides and ordered by size. Source data are provided as a Source data file.