Fig. 1: Association of MTBP and phospho-RecQL4 with pre-RCs at baseline and dormant replication origins.

A Baseline and dormant origins. Top, experimental procedure. SIRT1 deficient (SIRT1^Null) HCT116 cells were complemented with either WT-SIRT1 (WT^SIRT1) or H363Y-SIRT1 (an inactive mutant; Mut^SIRT1) cDNA¹⁵. Replication origins were mapped using nascent strand sequencing (NS-seq)¹⁵. Bottom, a genome browser view of NS-seq coverage across a representative genomic region on chromosome 1. Baseline origins (black rectangles) are defined as active in both WT^SIRT1 and Mut^SIRT1 cells, whereas dormant origins (dashed black rectangles) are active only in Mut^SIRT1 cells. Pre-RNase treatment serves as a control, as RNA primer removal allows lambda exonuclease to digest nascent DNA, eliminating origin peaks. B Nascent strand abundance (initiation) at baseline and dormant origins in cells harboring WT^SIRT1 (WT) or Mut^SIRT1 (Mut). C HCT116 cells containing auxin – inducible degron MCM2 (MCM2-mAID) were complemented with either intact (WT) or S139A substituted MCM2¹⁵. IAA (auxin, 500 μM for 16 h) depleted the endogenous MCM2-mAID. Complementation with MCM2-S139A, but not WT MCM2, inhibited replication initiation¹⁵ (Supplementary Fig. 1E). D The abundance of pMCM2-S139, MCM2, Treslin, MTBP and pRecQL4-S89 in whole cell (WCE) and chromatin extracts (ChrE) from HCT116 cells harboring MCM2-mAID complemented with MCM2-WT and MCM2-S139A with and without IAA. Representative of three independent replicates. The expression of MCM2-WT and MCM2-S139A was similar and comparable to the endogenous protein. E, F Binding sites of pMCM2-S139, MTBP, Treslin, RecQL4, and pRecQL4-S89 mapped by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) in WT^SIRT1 and Mut^SIRT1 cells. pMCM2-S139 binding was measured in G1-synchronized cells, whereas binding of MTBP, Treslin, RecQL4, and pRecQL4-S89 was measured in asynchronous cells. E ChIP-seq coverage at the genomic region depicted in panel (A). From top, baseline and dormant origins; averaged ChIP-seq coverage (from two biological replicates) for MTBP, Treslin, pRecQL4-S89, pMCM2-S139, pMCM2-S139, RecQL4 and input controls from WT^SIRT1 and Mut^SIRT1. F Heatmaps depicting chromatin binding at baseline and dormant replication origins in HCT116 cells harboring WT^SIRT1 or Mut^SIRT1 as measured as in panel (E). Baseline and dormant origins were stratified based on replication initiation activity as depicted in panels (A) and (B). G Quantification of chromatin binding patterns shown in panel (F) (above). Source Data are provided as a Source Data file.