Polθ is phosphorylated by Polo-like kinase 1 (PLK1) to enable repair of DNA double strand breaks in mitosis

Background

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that if left unrepaired can lead to severe genomic instability or cell death. Our cells have evolved several repair mechanisms that deal with this type of damage, which are regulated across the cell cycle, by the chromatin landscape, and the configuration of broken ends (Scully et al., 2019).

One such pathway is Theta-Mediated End Joining (TMEJ) and its activity has been observed in several organisms including Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Mus Musculus and Homo sapiens (Schimmel et al., 2019). This repair mechanism is by definition mutagenic as it relies on DNA polymerase theta (Polϴ, encoded by the gene POLQ) which uses the microhomology of the opposing 3′ single-stranded DNA to anneal and repair chromosomal breaks. This leads to specific genomic “scars”, such as deletions and templated insertions (Schimmel et al., 2017). Thus, the use of TMEJ strongly jeopardizes the integrity of our genome and may promote the onset of pathologies such as cancer.

A key question in the field is how cells can benefit from the use of such a repair mechanism? Interestingly, Polϴ activity protects from gross chromosomal rearrangements such as large DNA deletions (Roerink et al., 2014; van Schendel et al., 2016). In addition, POLQ deficiency increases sensitivity to ionizing radiation (Higgins et al., 2010) and micronuclei formation (Shima et al., 2004), implying that TMEJ supports the repair of DNA breaks that other pathways – such as non-homologous end joining (NHEJ) and homologous recombination (HR) – cannot support. In line with this hypothesis, it was shown that TMEJ is more active toward DSBs occurring in specific heterochromatic regions and blunt broken ends (Schep et al., 2021; Schimmel et al., 2017). On top of that, Polϴ deficiency confers synthetic lethality with a deficiency of genes encoding HR factors (Ceccaldi et al., 2015; Mateos-Gomez et al., 2015) and the Shieldin2 gene involved in NHEJ (Zatreanu et al., 2021b), arguing that TMEJ is a pathway operating in parallel with NHEJ and HR.

An alternative hypothesis would be that TMEJ remains operational when NHEJ and HR are not. Indeed, while the NHEJ and HR repair pathways are highly active during interphase, they are blocked during mitosis (Heijink et al., 2013; Orthwein et al., 2014). The active inhibition of NHEJ and HR during mitosis is orchestrated by the kinases Polo-like kinase 1 (PLK1) and Cyclin dependent kinase 1 (CDK1) which target and inhibit essential factors of these repair pathways such as 53BP1 and CtIP (Benada et al., 2015; Wang et al., 2018).

Conversely, the activity of the TMEJ repair pathway during mitosis has so far been little studied. Interestingly, a recent study has shown that synthetic DSBs incubated with mitotic cell extracts pull-down Polϴ, suggesting a role for TMEJ during this phase of the cell cycle (Heijink et al., 2022).

In this preprint (Gelot Camille et al., 2023), the Ceccaldi lab unveils that the kinase PLK1 licenses TMEJ-mediated DSB repair during mitosis by phosphorylating Polϴ thereby ensuring genome stability.

 

Key findings

Polϴ is recruited to mitotic DNA breaks

With the aim of characterizing the dynamics of Polϴ in response to DNA damage and its modes of regulation, the authors studied the foci formation of fluorescently-tagged Polϴ (endogenous or exogenous) in response to various genotoxic stresses that induce DNA double-strand breaks.

The authors found that Polϴ forms foci during S phase of the cell cycle in response to ionizing radiation (IR). Remarkably, cells in G2 or in mitosis (Fig1A) that had been exposed to replication stress during S phase showed an increase in Polϴ foci, suggesting that Polϴ binds DNA breaks during S phase and persists on those that are unrepaired and transmitted into mitosis. In addition, mitotic cells that were exposed to ionizing radiation displayed Polϴ foci, indicating that binding of Polϴ to mitotic DSBs is not restricted to breaks induced during S phase.

 

Polϴ repairs mitotic DNA breaks

Strikingly, the recruitment of Polϴ to mitotic DSBs was shown to rely on ATM, MDC1 and TOPBP1; which are factors described to accumulate in mitosis in response to DNA damage while NHEJ and HR factors do not. Furthermore, the authors demonstrated that, together with ATM, Polϴ regulates the mitotic checkpoint activation and is crucial to resolve Cas9- and IR-induced DSBs in mitosis. Finally, immunofluorescence colocalization experiments strongly suggested that Polϴ is not involved in mitotic DNA synthesis (MiDas). Collectively, the authors reveal that Polϴ defines an active and major pathway of DSB repair during mitosis.

 

PLK1 drives TMEJ in mitosis

The authors further investigated the mechanism by which Polϴ is regulated during the repair of DSBs in mitosis. Using several orthogonal approaches, they revealed that Polo-Like Kinase 1 (PLK1) directly phosphorylates Polϴ on several serine residues. Remarkably, phosphorylation of these serine residues and the activity of PLK1 are required for both recruitment of Polϴ to the sites of DNA damage in mitosis (Fig1 B) and the repair of DNA breaks. Thus, the authors unveiled that PLK1 licenses TMEJ-mediated DSB repair in mitosis while it inhibits both NHEJ and HR.

 

Figure 1. PLK1-dependent Polϴ phosphorylation drives its recruitment to mitotic DNA breaks (with panels from (Gelot Camille et al., 2023)). (A) Representative micrographs of immunofluorescence showing GFP-tagged Polϴ foci (green) localizing at mitotic DNA breaks (gammaH2A.X, Red) in human cells following replicative stress induced by the inhibition of ATR (ATRi). (B) Representative micrographs of either WT GFP-Polϴ or the GFP-PhD-Polϴ mutant in interphasic and mitotic cells. While the wild type of Poltheta (WT- Polϴ) is recruited to DNA breaks in interphase and mitosis, the mutant form that cannot be phosphorylated by PLK1 (PhD-Polϴ) is not recruited to mitotic DNA breaks.

 

TMEJ in mitosis safeguards genome stability

In addition, and in a remarkable way, the authors showed that PLK1-dependent TMEJ in mitosis prevents micronuclei formation, larger deletions, and that the synthetic lethality between POLQ and BRCA2 genes results from unrepaired DSB accumulation in mitosis. Thereby, the authors unveiled that Polϴ-mediated double-strand break repair protects against genomic instability during mitosis and that this pathway is required for the survival of cells deficient in HR.

 

Conclusion

By showing that Polϴ repairs DSBs during mitosis, the authors have redefined the physiological function of TMEJ. Often considered as an alternative and minor DNA repair pathway, the authors now reveal that DSB repair by Polϴ is the major pathway during mitosis. Despite its mutagenic nature, this pathway can be used by cells to take over mitotic or unrepaired DSBs from S/G2 phases to prevent chromosome rearrangements and to survive at the expense of small deletions.

 

References

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What I like about this preprint

Using multiple approaches, the authors defined a new mechanism for the regulation of DSB repair during mitosis by Polϴ. By showing that the survival of cells deficient in homologous recombination depends on this pathway, the authors open up new therapeutic perspectives for the use of PARP inhibitors in the treatment of tumors deficient for the BRCA1/2 genes, in particular by aiming at new targets in order to potentiate the efficacy of anticancer treatments.

 

Questions for the authors

Q1: In your study, you demonstrate that the conservation of PLK1-targeted Polϴ residues is required for the survival of BRCA2-deficient cells. This and other results thus demonstrate that synthetic lethality following BRCA2 and Polϴ depletion is due to a defect in DSB repair during mitosis. Naively, one might think that PLK1 deficiency combined with BRCA2 deficiency would also be lethal to cells. However, as mentioned in your study, PLK1 also inhibits NHEJ and HR in mitosis. So, do you know whether PLK1 inhibition or deficiency would mimic Polϴ deficiency in BRCA2-deficient cells?

Q2: In your study, you also show that Polϴ forms foci during S phase in wild-type cells (proficient for HR). In addition, Polϴ recruitment in S phase depends on several factors that regulate HR. Although HR and TMEJ are mutually exclusive repair pathways, it is tempting to think that these factors may ultimately activate TMEJ in the event that HR cannot proceed. Could you share your thoughts on these intriguing results? Do you think that factors involved in the SSA repair pathway could also stimulate the formation of Polϴ foci to DNA damage?

 

Acknowledgements

I thank Dr. Robin van Schendel (LUMC, Leiden, the Netherlands) and Dr. Reinier Prosée (Prelights team) for critical reading of the manuscript.

Tags: dna double-strand break, dna polymerase theta, mitosis, plk1

doi: https://doi.org/10.1242/prelights.34811

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