Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of "stressed" replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. ChIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late.
Bibliographical noteThis is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply, 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.
Acknowledgements & Funding: This research was supported, in part, by the Intramural Research Program of the NIH, National Institute on Aging, United States (Z01-AG000746-08). G.S.M. and G.S.S. received funding from the CR-UK Program grant (C17183/A23303), and J.J.R. was supported by the University of Birmingham. This work utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). We thank Cuong Nguyen and Tonya Wallace of the NIA Flow Cytometry Unit for expert support and guidance, Supriyo De and William Wood for DNA sequencing and data extraction, Dr. Florencia Pratto and Dr. Amit Singh for data analysis, and Dr. Yie Liu, Dr. Rafael D. Camerini-Otero, and Dr. Weidong Wang for helpful suggestions and discussions.
- Cell Cycle Proteins/metabolism
- Chromatin Immunoprecipitation Sequencing
- DNA Helicases/metabolism
- DNA Replication Timing
- HeLa Cells
- Nuclear Proteins/metabolism
- S Phase