Abstract
During S phase, replication forks can encounter several obstacles that lead to fork stalling, which if persistent might result in fork collapse. To avoid this collapse and to preserve the competence to restart, cells have developed mechanisms that maintain fork stability upon replication stress. In this study, we aimed to understand the mechanisms involved in fork stability maintenance in non-transformed human cells by performing an isolation of proteins on nascent DNA-mass spectrometry analysis in hTERT-RPE cells under different replication stress conditions. Our results show that acute hydroxyurea-induced replication blockade causes the accumulation of large amounts of single-stranded DNA at the fork. Remarkably, this results in the disengagement of replisome components from nascent DNA without compromising fork restart. Notably, Cdc45-MCM-GINS helicase maintains its integrity and replisome components remain associated with chromatin upon acute hydroxyurea treatment, whereas replisome stability is lost upon a sustained replication stress that compromises the competence to restart.
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Abbreviations
- CMG:
-
Cdc45-MCM-GINS
- DSBs:
-
Double-strand breaks
- HR:
-
Homologous recombination
- BIR:
-
Break-induced replication
- ssDNA:
-
Single-stranded DNA
- HU:
-
Hydroxyurea
- FBS:
-
Fetal bovine serum
- PFA:
-
Paraformaldehyde
- RT:
-
Room temperature
- PIC:
-
Protease inhibitor cocktail
- PI:
-
Propidium iodide
- iPOND:
-
Isolation of proteins on nascent DNA
- MS:
-
Mass spectrometry
- WB:
-
Western blot
- QIBC:
-
Quantitative image-based cytometry
- RPC:
-
Replication pausing complex
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Acknowledgements
We thank Dr. Surrallés for Fen1, Dr. Mendéz for MCM3, and Dr. Stracker for SMC1 and Pan-MCM antibodies. We also thank the members of our laboratory for their discussion and the advanced optical microscopy unit of the CCiT-UB for its technical assistance. This work was supported by the grants from the Ministerio de Economia y Competitividad (SAF2013-42742-R, SAF2016-76239-R) for N.A; an FPI fellowship from the Ministerio de Ciencia e Innovación for A.E. and A.Ll.; and an FI fellowship from the Generalitat de Catalunya for S.F.
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Ercilla, A., Feu, S., Aranda, S. et al. Acute hydroxyurea-induced replication blockade results in replisome components disengagement from nascent DNA without causing fork collapse. Cell. Mol. Life Sci. 77, 735–749 (2020). https://doi.org/10.1007/s00018-019-03206-1
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DOI: https://doi.org/10.1007/s00018-019-03206-1