Search

Background : It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the genomic location of origin clusters has not been documented. Methods : We synchronised human U2OS by thymidine block and release followed by L-mimosine block and release to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped onto the human genome. Results : The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters had a statistical preference for being activated in sequence across a group, consistent with the 'domino' model of replication focus activation order observed by microscopy. Conclusions : We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated., Competing Interests: No competing interests were disclosed., (Copyright: © 2023 da Costa-Nunes JA et al.)

Mammalian chromosomes are organized at different length scales within the cell nucleus. Topologically Associating Domains (TADs) are structural units of 3D genome organization with functions in gene regulation, DNA replication, recombination and repair. Whereas TADs were initially interpreted as insulated domains, recent studies are revealing that these domains should be interpreted as dynamic collections of actively extruding loops. This process of loop extrusion is subsequently blocked at dedicated TAD boundaries, thereby promoting intra-domain interactions over their surroundings. In this review, we discuss how mammalian TAD structure can emerge from this dynamic process and we discuss recent evidence that TAD boundaries can have regulatory functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)