Acute hydroxyurea-induced replication blockade results in replisome components disengagement from nascent DNA without causing fork collapse
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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.
KeywordsiPOND Replication fork stability CMG Replication stress
Fetal bovine serum
Protease inhibitor cocktail
Isolation of proteins on nascent DNA
Quantitative image-based cytometry
Replication pausing complex
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.
Compliance with ethical standards
Conflict of interest
The authors declare no competing interests.
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