Mre11 exonuclease activity promotes irreversible mitotic progression under replication stress.

Yoshitami Hashimoto,Hirofumi Tanaka

doi:10.26508/lsa.202101249

Yoshitami Hashimoto, Hirofumi Tanaka

Open Access

https://doi.org/10.26508/lsa.202101249

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Abstract

Mre11 is a versatile exo-/endonuclease involved in multiple aspects of DNA replication and repair, such as DSB end processing and checkpoint activation. We previously demonstrated that forced mitotic entry drives replisome disassembly at stalled replication forks in Xenopus egg extracts. Here, we examined the effects of various chemical inhibitors using this system and discovered a novel role of Mre11 exonuclease activity in promoting mitotic entry under replication stress. Mre11 activity was necessary for the initial progression of mitotic entry in the presence of stalled forks but unnecessary in the absence of stalled forks or after mitotic entry. In the absence of Mre11 activity, mitotic CDK was inactivated by Wee1/Myt1-dependent phosphorylation, causing mitotic exit. An inhibitor of Wee1/Myt1 or a nonphosphorylatable CDK1 mutant was able to partially bypass the requirement of Mre11 for mitotic entry. These results suggest that Mre11 exonuclease activity facilitates the processing of stalled replication forks upon mitotic entry, which attenuates the inhibitory pathways of mitotic CDK activation, leading to irreversible mitotic progression and replisome disassembly.

Highlights

Eukaryotic DNA replication is initiated at replication origins during the S-phase of the cell cycle, forming replication forks to which the replisome, a multi-protein complex, binds and conducts DNA replication
When the first incubation was extended to 90 min and performed without aphidicolin to allow for the completion of DNA replication, mirin did not inhibit mitotic entry, and a small amount of residual replisome components were dissociated from chromatin (Fig 1E, lanes 1, 2, and 4)
Mirin did not inhibit interphase replisome disassembly in the absence of aphidicolin, as opposed to NMS873 which caused persistent chromatin binding of claspin, Psf2, and poly-ubiquitylated Mcm7 at later time points (Fig 2A). These results suggest that the exonuclease activity of Mre11 is required for mitotic entry and mitotic replisome disassembly (MRD) in the presence of stalled replication forks but not when DNA replication is nearly complete and without exogenous inhibition

Summary

Introduction

Eukaryotic DNA replication is initiated at replication origins during the S-phase of the cell cycle, forming replication forks to which the replisome, a multi-protein complex, binds and conducts DNA replication. Replication forks can often encounter obstacles for their progression, such as DNA lesions, nucleotide reduction, secondary DNA structures, DNA–RNA hybrids, and transcriptional machinery (Zeman & Cimprich, 2014; Gaillard et al, 2015). These replication stress factors cause stalled replication forks, which are stabilized for replication restart until the stress is relieved. Reversed forks are believed to contribute to fork stabilization and repair, and stalled forks (or reversed forks) have been shown to activate ATRChk checkpoint signaling to down-regulate CDK1/2 activities via Cdc inactivation, suppressing de novo initiation of DNA replication and cell cycle progression into G2/M phases (Saldivar et al, 2017). Cdc usually activates Cdk1/2 during S to G2/M phase transition to drive replication initiation and cell cycle progression by removing Wee1/Myt kinase–mediated inhibitory phosphorylation of Thr and Tyr (Sørensen & Syljuasen, 2012; Crncec & Hochegger, 2019; Elbæk et al, 2020)

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