POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells

Beláň, Ondrej; Sebald, Marie; Adamowicz, Marek; Anand, Roopesh; Vančevska, Aleksandra; Neves, Joana; Grinkevich, Vera; Hewitt, Graeme; Segura-Bayona, Sandra; Bellelli, Roberto; Robinson, Helen M.R.; Higgins, Geoff S.; Smith, Graeme C.M.; West, Stephen C.; Rueda, David; Boulton, Simon J.

doi:10.1016/j.molcel.2022.11.008

Cited by 61 publications

(48 citation statements)

References 65 publications

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“…It is also plausible that a RHINO-TOPBP1 interaction in mitosis could stabilize RHINO at break sites. Finally, our data do not rule out the possibility of additional roles for Polθ in filling ssDNA gaps accumulating during S phase and persisting through mitosis (Belan et al , 2022; Mann et al , 2022; Roerink et al , 2014; Schrempf et al , 2022). In conclusion, our study provides evidence suggesting robust MMEJ activity in mitosis that may account for the synthetic lethal interaction between Polθ and BRCA2.…”

Section: Discussioncontrasting

confidence: 80%

RHINO restricts MMEJ activity to mitosis

Sfeir

Brambati

Sacco

et al. 2023

Preprint

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DNA double-strand breaks (DSBs) are toxic lesions that can lead to genome instability if not properly repaired. Breaks incurred in G1 phase of the cell cycle are predominantly fixed by non-homologous end-joining (NHEJ), while homologous recombination (HR) is the primary repair pathway in S and G2. Microhomology-mediated end-joining (MMEJ) is intrinsically error-prone and considered a backup DSB repair pathway that becomes essential when HR and NHEJ are compromised. In this study, we uncover MMEJ as the major DSB repair pathway in M phase. Using CRISPR/Cas9-based synthetic lethal screens, we identify subunits of the 9-1-1 complex (RAD9A-HUS1-RAD1) and its interacting partner, RHINO, as critical MMEJ factors. Mechanistically, we show that the function of 9-1-1 and RHINO in MMEJ is inconsistent with their well-established role in ATR signaling. Instead, RHINO plays an unexpected and essential role in directing mutagenic repair to M phase by directly binding to Polymerase theta and promoting its recruitment to DSBs in mitosis. In addition, we provide evidence that mitotic MMEJ repairs persistent DNA damage that originates in S phase but is not repaired by HR. The latter findings could explain the synthetic lethal relationship between POLQ and BRCA1/2 and the synergistic effect of PolQ and PARP inhibitors. In summary, our study identifies MMEJ as the primary pathway for repairing DSBs during mitosis and highlights an unanticipated role for RHINO in directing mutagenic repair to M phase.

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Section: Discussioncontrasting

confidence: 80%

RHINO restricts MMEJ activity to mitosis

Sfeir

Brambati

Sacco

et al. 2023

Preprint

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“…Our study does not rule out a role for Polq in filling ssDNA gaps after replication, nor do we exclude the possibility of RHINO-independent MMEJ activity during S phase (65)(66)(67)(68). However, our findings provide evidence suggesting that robust MMEJ activity in mitosis accounts for the synthetic lethal interaction between Polq and BRCA2.…”

Section: Discussioncontrasting

confidence: 74%

RHINO directs MMEJ to repair DNA breaks in mitosis

Brambati

Sacco

Porcella

et al. 2023

Science

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Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the primary pathways for repairing DNA double-strand breaks (DSBs) during interphase, while microhomology-mediated end-joining (MMEJ) has been regarded as a backup mechanism. Through CRISPR/Cas9-based synthetic lethal screens, we identify subunits of the 9-1-1 complex (RAD9A-HUS1-RAD1) and its interacting partner, RHINO, as crucial MMEJ factors. We uncover an unexpected function for RHINO in restricting MMEJ to mitosis. RHINO accumulates in M phase, undergoes PLK1 phosphorylation, and interacts with polymerase theta (Polθ), enabling its recruitment to DSBs for subsequent repair. Additionally, we provide evidence that MMEJ activity in mitosis repairs persistent DSBs originating in S phase. Our findings offer insights into the synthetic lethal relationship between POLQ and BRCA1/2 and the synergistic effect of Polθ and PARP inhibitors.

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“…We believe this provides an opportunity to target both pathways simultaneously to improve tumour eradication and reduce risk of resistance to either therapy individually. Next to a role in DSB repair, POLQ has recently been described to be involved in ssDNA gap fill-in (Belan et al, 2022; Mann et al, 2022; Schrempf et al, 2022). However, it is currently unclear how the two different roles of POLQ contribute to its essential role in BRCA-deficient cells.…”

Section: Discussionmentioning

confidence: 99%

“…This implies that DSB repair by alt-EJ or SSA is essential for survival of HR-deficient cells (Ceccaldi et al, 2015; Feng et al, 2011; Lok et al, 2013; Mateos-Gomez et al, 2015). However, recent studies suggested that increased ssDNA gap formation in absence of PolQ may underlie the synthetic lethality with BRCA-deficiency (Belan et al, 2022; Mann et al, 2022; Schrempf et al, 2022). BRCA-deficient cells are prone to accumulate ssDNA gaps, and toxic levels of these lesion are associated with sensitivity to chemotherapy and PARPi (Cong et al, 2021; Paes Dias et al, 2021; Panzarino et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

Kooij

Schreuder

Pavani

et al. 2023

Preprint

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Deficiency for the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR) leads to chromosomal instability and diseases such as cancer. Yet, defective HR also results in vulnerabilities that can be exploited for targeted therapy. Here, we identify such a vulnerability and show that BRCA1-deficient cells are dependent on the long-range end-resection factor EXO1 for survival. EXO1 loss results in DNA replication-induced lesions decorated by poly(ADP-ribose)-chains. In cells that lack both BRCA1 and EXO1, this is accompanied by unresolved DSBs due to impaired single-strand annealing (SSA), a DSB repair process that requires the activity of both proteins. In contrast, BRCA2-deficient cells have increased SSA, also in the absence of EXO1, and hence are not dependent on EXO1 for survival. In agreement with our mechanistic data, BRCA1-mutated tumours have elevated EXO1 expression and contain more genomic signatures of SSA compared to BRCA1-proficient tumours. Collectively, our data indicate that EXO1 is a promising novel target for treatment of BRCA1-deficient tumours.

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POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells

Cited by 61 publications

References 65 publications

RHINO restricts MMEJ activity to mitosis

RHINO restricts MMEJ activity to mitosis

RHINO directs MMEJ to repair DNA breaks in mitosis

EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

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