Active transcription regulates ORC/MCM distribution whereas replication timing correlates with ORC density in human cells

Abstract

42Eukaryotic replication initiates during S phase from origins that have been licensed in 43 the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors 44Orc2, Orc3, Mcm3, and Mcm7 with replication initiation events obtained by Okazaki 45 fragment sequencing. We demonstrate that MCM is displaced from early replicating, 46 actively transcribed gene bodies, while ORC is mainly enriched at active TSS. Late 47Mammalian DNA replication is a highly orchestrated process ensuring the 55 exact inheritance of genomes of tens to thousands of million base pairs in size. In 56 human cells, replication initiates from 30,000 -50,000 replication origins per cell 1,2 . 57 Origins are not activated synchronously but are organized into individual replication 58 timing domains (RTDs), which replicate in a timely coordinated and reproducible 59 order from early to late in S phase 3,4 . The replication cascade or domino model 60 proposes that within one RTD, replication first initiates at the most efficient origins 61 and then spreads to less efficient origins. RTDs are separated by timing transition 62 regions and it is debated whether replication spreading is blocked at these regions 5,6 . 63The establishment of replication competence occurs in late mitosis and during 64 the G1 phase of the cell cycle 7 . The first step is the cell-cycle dependent assembly of 65 the evolutionary conserved origin recognition complex (ORC) to origins 8,9 . ORC and 66 two chaperones, Cdt1 and Cdc6, cooperatively load minichromosome maintenance 67 (MCM) complexes as double hexamers 10-12 . The MCM complex is the central unit of 68 the replicative helicase. The resulting multi-subunit complex is termed pre-replicative 69 complex (pre-RC). A single ORC loads multiple MCM helicases, which are 70 translocated from their original loading site, but no ORC, neither Cdc6, nor Cdt1 are 71 required for origin activation 13,14 . 72Replication origins are defined functionally. In the unicellular S. cerevisiae, 73 replication origins are genetically characterized by the ARS consensus sequence 15 . In 74 multicellular organisms, replication initiates from flexible locations and no common 75 consensus element for origin selection and activation has yet been identified. It is 76 generally believed that chromatin features including histone modifications, 77 nucleosome dynamics and DNA modifications contribute to origin specification 1,16,17 , 78 including H4K20me3 that supports the licensing of a subset of late replicating origins in heterochromatin 18 . Thus, changing environmental conditions, DNA damage and the 80 development status of each cell are integrated into highly dynamic local chromatin 81 profiles, which influence the plasticity of origin selection 1 . 82 Different approaches have been developed to characterize mammalian 83 replication initiation by single-molecule visualization (DNA combing) or sequencing 84 of purified initiation products (short nascent strands (SNS-seq), initiation site 85 sequencing (INI-seq) replicatio...