Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones

Nina Kirstein,Elisabeth Kremmer,Stefan Krebs,Olivier Hyrien,Wolfgang Hammerschmidt,Xia Wu,Helmut Blum,Ina M Vorberg,Benjamin Audit,Alexander Buschle,Aloys Schepers,Laurent Lacroix

doi:10.7554/elife.62161

Abstract

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing, and fork directionality profiles obtained by RNA-seq, Repli-seq, and OK-seq. Both, the origin recognition complex (ORC) and the minichromosome maintenance complex (MCM) are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating regions, and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late-replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.

Highlights

In human cells, DNA replication initiates from 20,000 to 50,000 replication origins selected from a five- to tenfold excess of potential or ‘licensed’ origins (Moiseeva and Bakkenist, 2018; Papior et al, 2012)
origin recognition complex (ORC)/maintenance complex (MCM) were enriched at the H4K20me3-high subgroup compared to the H4K20me3-low subgroup (Figure 5f). These results suggest that the presence of H4K20me3 at transcriptionally independent, non-genic ascending segments (ASs) may contribute at the origin-licensing step to specifying these regions as highly efficient ‘master’ initiation zones (Ma-IZs) in late-replicating DNA
We further show that subsets of nongenic ASs and randomly replicating gene deserts are enriched in H4K20me3, which helps recruiting Origin recognition complex/minichromosome maintenance complex (ORC/MCM) to these late-replicating segments

Summary

Introduction

DNA replication initiates from 20,000 to 50,000 replication origins selected from a five- to tenfold excess of potential or ‘licensed’ origins (Moiseeva and Bakkenist, 2018; Papior et al, 2012). Called pre-replicative complex (pre-RC) formation, occurs in late mitosis and during the G1 phase of the cell cycle. During this step, the origin recognition complex (ORC) binds DNA and, together with Cdt and Cdc, loads minichromosome maintenance complexes (MCM), the core motor of the replicative helicase, as inactive head-to-head double hexamers (MCM-DHs) around double-stranded DNA MCM-DHs that do not initiate replication are dislodged from DNA during replication

Results

Discussion

Conclusion