Fig. 5

Replication initiation occurs at the transcription barrier. a Schematic of dCas9-gRNA-mediated transcription blockade. Four gRNAs were designed to bind the non-template strand at the fourth exon of CMIP. The orange arrow indicates the transcribed region and the red stop sign illustrates the dCas9-induced transcription blockade. b, d The mean of EdU/HU normalized read density (line) with the standard error (shadow) of three biological replicates near the dCas9/gRNA binding sites on CMIP in early S phase (b) or G1-phase (d) K562 cells treated with CMIP (red) or scrambled (cyan) gRNAs. gRNA-CMIP and gRNA-scramble indicate that cells were treated with dCas9/gRNA targeting CMIP or a control region, respectively. The regions between two dashed lines around 0 highlight the four binding sites of CMIP gRNAs. The 10-kb window of dCas9 binding sites is tiled by 1-kb bins (sliding by 200 bp). The read count per kilobase (RPK) was calculated within each 1-kb bin and normalized by the RPK of the B compartments from each biological replicate, defining the RPK ratio (see “Methods” for details). c, e The levels of EdU/HU read density upstream (− 2 k) or downstream (2 k) of the gRNA-CMIP binding sites in early S phase (c) or G1 phase (e) K562 cells. The read density was calculated in 2-kb bins. Student’s t-test, p = 0.025; *, p < 0.05; n.s., no significance. f ChIP-qPCR showing the distribution of MCM5 surrounding dCas9-binding sites in CMIP. A biological replicate of MCM5 ChIP-qPCR and the other two biological replicates are shown in Additional file 1: Figure S6i. t-test, mean ± SD. “Up” or “down” indicates the upstream or downstream non-transcribed regions of CMIP, respectively. The other five positions are the same as those shown in b and d. g Schematic of the transcription bulldozing model. Left: in the G1 phase, MCM double hexamers (MCM-DHs) loaded by ORC in transcribed regions are driven along gene bodies by RNA polymerase II to downstream non-transcribed regions. Accumulated MCM-DHs in non-transcribed regions initiate DNA replication in the early S phase. Right: when transcription is inhibited, MCM-DHs accumulate around the ORC binding site and initiate DNA replication at gene bodies