Regulated TRESLIN-MTBP loading governs initiation zones and replication timing in human DNA replication [LD-OK-seq]

Replication origins in human cells form clusters ranging from tens to hundreds of kilobases called initiation zones (IZs), typically located in intergenic regions between active genes. On a larger megabase scale, chromosomes replicate in a temporally defined replication timing (RT) pattern, where euchromatic regions replicate early, and heterochromatic regions replicate late. Although the stochastic model of IZ firing with a temporally regulated limiting factor can explain RT formation, this limiting factor in human cells remains unclear. To investigate the relationship between IZ and RT, we mapped the temporal firing pattern of IZs and examined the genome-wide distributions of replication licensing and firing factors in human cells. We identified TRESLIN-MTBP as the key limiting firing factor for replication initiation. Its loading onto phosphorylated MCM2–7 double hexamer (MCM-DH) is controlled by the opposing phosphorylation events on MCM-DH by Dbf4-dependent kinase and RIF1-Protein Phosphatase 1, which ultimately determine IZs and establish RT. Overall design: Ligase-depleted Okazaki-sequencing (LD-OK-seq) was performed using HCT116 cells. LD-OK-seq experiments were conducted under both asynchronous and synchronous conditions. Synchronous LD-OK-seq was carried out at six time points across the S phase. The same LD-OK-seq experiments were also performed using HCT116 cells with RIF1 knockout (RIF1-KO). Additionally, RIF1-KO cells with reintroduction of either wild-type RIF1 (WT-RIF1) or a RIF1-ppC mutant were used. Furthermore, cells overexpressing TRESLIN-MTBP were also subjected to LD-OK-seq.