Journal of Biological Chemistry
Volume 296, January–June 2021, 100033
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Research Article
Ongoing replication forks delay the nuclear envelope breakdown upon mitotic entry

https://doi.org/10.1074/jbc.RA120.015142Get rights and content
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DNA replication is a major contributor to genomic instability, and protection against DNA replication perturbation is essential for normal cell division. Certain types of replication stress agents, such as aphidicolin and hydroxyurea, have been shown to cause reversible replication fork stalling, wherein replisome complexes are stably maintained with competence to restart in the S phase of the cell cycle. If these stalled forks persist into the M phase without a replication restart, replisomes are disassembled in a p97-dependent pathway and under-replicated DNA is subjected to mitotic DNA repair synthesis. Here, using Xenopus egg extracts, we investigated the consequences that arise when stalled forks are released simultaneously with the induction of mitosis. Ara-cytidine-5′-triphosphate–induced stalled forks were able to restart with the addition of excess dCTP during early mitosis before the nuclear envelope breakdown (NEB). However, stalled forks could no longer restart efficiently after the NEB. Although replisome complexes were finally disassembled in a p97-dependent manner during mitotic progression whether or not fork stalling was relieved, the timing of the NEB was delayed with the ongoing forks, rather than the stalled forks, and the delay was dependent on Wee1/Myt1 kinase activities. Thus, ongoing DNA replication was found to be directly linked to the regulation of Wee1/Myt1 kinases to modulate cyclin-dependent kinase activities because of which DNA replication and mitosis occur in a mutually exclusive and sequential manner.

Keywords

replisome
replication forks
Xenopus egg extract
nuclear envelope breakdown (NEB)
CDK
Wee1/Myt1

Abbreviations

Ara-CTP
Ara-cytidine-5′-triphosphate
CDKs
cyclin-dependent kinases
ex-dCTP
excessive amounts of dCTP
M-CDK
M-phase CDK
M-extract
M-phase extract
MiDAS
mitotic DNA synthesis
NE
nuclear envelope
NEB
nuclear envelope breakdown
p97i
p97 inhibitor
PD
PD166285
Pre-RCs
prereplicative complex
S-extract
S-phase extract

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