The RAD51 recombinase protects mitotic chromatin in human cells

Isabel E Wassing,Christine Ralf,Andrew Bassett,Fumiko Esashi,Emily Graham,Xanita Saayman,Lucia Rampazzo

doi:10.1038/s41467-021-25643-y

Isabel E Wassing, Christine Ralf + Show 5 more

Open Access

https://doi.org/10.1038/s41467-021-25643-y

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Abstract

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.

Highlights

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells
In proof-of-principle experiments (Supplementary Fig. 1b and c), we addressed the possibility that EdU incorporation in late G2 contributes to the resultant EdU foci detected in mitotically collected cells by administrating the EdU pulse 30 min prior to mitotic release (m-R) with or without a subsequent chasing with unlabelled thymidine
Given that the thymidine chase outcompetes the incorporation of trace EdU amounts remaining inside of cells after m-R, this observation demonstrates that the EdU signal observed in G2 pulse-labelled cells mostly represents EdU incorporation after m-R, and that negligible EdU incorporation occurs during the G2-pulse itself

Summary

Introduction

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. Our previous studies uncovered a separate mechanism that enables RAD51 recruitment to damaged DNA independently of BRCA2 chromatin enrichment[17,18] This pathway is triggered by a key mitotic regulator, Polo-like kinase 1 (PLK1), which phosphorylates RAD51 at serine 14 (S14) shortly after DNA damage and in later phases of the cell cycle. This event subsequently provokes RAD51 phosphorylation by the acidophilic casein kinase 2 (CK2) at threonine 13 (T13), which mediates a direct interaction between RAD51 and the NBS1 component of the MRN complex.

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