Single-cell replication profiling to measure stochastic variation in mammalian replication timing

Vishnu Dileep,David M Gilbert

doi:10.1038/s41467-017-02800-w

Vishnu Dileep, David M Gilbert

Open Access

https://doi.org/10.1038/s41467-017-02800-w

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Journal: Nature Communications	Publication Date: Jan 30, 2018
Citations: 85	License type: open-access

Affiliation: Florida State University

Abstract

Mammalian DNA replication is regulated via multi-replicon segments that replicate in a defined temporal order during S-phase. Further, early/late replication of RDs corresponds to active/inactive chromatin interaction compartments. Although replication origins are selected stochastically, variation in replication timing is poorly understood. Here we devise a strategy to measure variation in replication timing using DNA copy number in single mouse embryonic stem cells. We find that borders between replicated and unreplicated DNA are highly conserved between cells, demarcating active and inactive compartments of the nucleus. Fifty percent of replication events deviated from their average replication time by ± 15% of S phase. This degree of variation is similar between cells, between homologs within cells and between all domains genomewide, regardless of their replication timing. These results demonstrate that stochastic variation in replication timing is independent of elements that dictate timing or extrinsic environmental variation.

Highlights

Mammalian DNA replication is regulated via multi-replicon segments that replicate in a defined temporal order during S-phase
Single DNA molecule studies have demonstrated that replication origins are selected stochastically, such that each cell is using a different cohort of origins to replicate their genome[3,4,5,6,7,8], replication timing is regulated independently of origin selection[9], and evidence suggests that replication timing is conserved in consecutive cell cycles[10,11,12]
These results demonstrate that stochastic variation in replication timing is independent of extrinsic environmental factors as well as the mechanisms controlling the temporal order of replication

Summary

Introduction

Mammalian DNA replication is regulated via multi-replicon segments that replicate in a defined temporal order during S-phase. We use DNA copy number variation (CNV) to measure replication timing in single cells at different stages in S phase.

Results

Conclusion