The location and development of Replicon Cluster Domains in early replicating DNA [version 2; peer review: 2 approved, 1 approved with reservations]
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 cluste...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Wellcome
2023-08-01
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| Series: | Wellcome Open Research |
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| Online Access: | https://wellcomeopenresearch.org/articles/8-158/v2 |
| _version_ | 1797690293354496000 |
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| author | Marek Gierlinski José A. da Costa-Nunes Emma J. Haagensen Takayo Sasaki J. Julian Blow David M. Gilbert |
| author_facet | Marek Gierlinski José A. da Costa-Nunes Emma J. Haagensen Takayo Sasaki J. Julian Blow David M. Gilbert |
| author_sort | Marek Gierlinski |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T01:57:27Z |
| format | Article |
| id | doaj.art-a76eca10627e41d8ae9b98b575ed5cfc |
| institution | Directory Open Access Journal |
| issn | 2398-502X |
| language | English |
| last_indexed | 2024-03-12T01:57:27Z |
| publishDate | 2023-08-01 |
| publisher | Wellcome |
| record_format | Article |
| series | Wellcome Open Research |
| spelling | doaj.art-a76eca10627e41d8ae9b98b575ed5cfc2023-09-08T01:00:00ZengWellcomeWellcome Open Research2398-502X2023-08-01822056The location and development of Replicon Cluster Domains in early replicating DNA [version 2; peer review: 2 approved, 1 approved with reservations]Marek Gierlinski0José A. da Costa-Nunes1Emma J. Haagensen2Takayo Sasaki3J. Julian Blow4https://orcid.org/0000-0002-9524-5849David M. Gilbert5https://orcid.org/0000-0001-8087-9737Data Analysis Group, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UKDivision of Molecular, Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UKDivision of Molecular, Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UKSan Diego Biomedical Research Institute, San Diego, California, CA 92121, USADivision of Molecular, Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UKSan Diego Biomedical Research Institute, San Diego, California, CA 92121, USABackground: 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.https://wellcomeopenresearch.org/articles/8-158/v2DNA replication S phase replicon clusters replication timing cell cycleeng |
| spellingShingle | Marek Gierlinski José A. da Costa-Nunes Emma J. Haagensen Takayo Sasaki J. Julian Blow David M. Gilbert The location and development of Replicon Cluster Domains in early replicating DNA [version 2; peer review: 2 approved, 1 approved with reservations] Wellcome Open Research DNA replication S phase replicon clusters replication timing cell cycle eng |
| title | The location and development of Replicon Cluster Domains in early replicating DNA [version 2; peer review: 2 approved, 1 approved with reservations] |
| title_full | The location and development of Replicon Cluster Domains in early replicating DNA [version 2; peer review: 2 approved, 1 approved with reservations] |
| title_fullStr | The location and development of Replicon Cluster Domains in early replicating DNA [version 2; peer review: 2 approved, 1 approved with reservations] |
| title_full_unstemmed | The location and development of Replicon Cluster Domains in early replicating DNA [version 2; peer review: 2 approved, 1 approved with reservations] |
| title_short | The location and development of Replicon Cluster Domains in early replicating DNA [version 2; peer review: 2 approved, 1 approved with reservations] |
| title_sort | location and development of replicon cluster domains in early replicating dna version 2 peer review 2 approved 1 approved with reservations |
| topic | DNA replication S phase replicon clusters replication timing cell cycle eng |
| url | https://wellcomeopenresearch.org/articles/8-158/v2 |
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