RHINO directs MMEJ to repair DNA breaks in mitosis

Abstract

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.

Broken chromosomes heading into mitosis: More than one way to patch a flat tire

“…This is because cells with interphase nuclear DNA damage typically utilize either HR or NHEJ before cell division. The MMEJ regulator POL THETA is frequently synthetic lethal in cells that are deficient for DNA repair by HR or NHEJ ( Brambati et al, 2023 ; Feng et al, 2019 ; Fleury et al, 2023 ), indicating a heightened role for MMEJ in cells deficient for interphase DDRs. Further, many cancer cells upregulate POL THETA expression ( Barszczewska-Pietraszek et al, 2022 ), which may indicate that cancers rewire DDRs to be more reliant on MMEJ.…”

“…Two very recent studies have shed light on molecular mechanisms, whereby Pol Theta is recruited to mitotic DSBs. In cultured human cells, Gelot et al (2023) identified a critical role for the mitotic kinase Polo-like kinase 1 (PLK1) in phosphorylating POL THETA ( Gelot et al, 2023 ), while Brambati et al (2023) identified the RHINO protein as a critical regulator of mitotic MMEJ ( Brambati et al, 2023 ).…”

“… Brambati et al (2023) used a synthetic lethality screen for cell survival factors in HR and NHEJ-deficient human cells, which lacked the repair factors BRCA2, LIG4, and TP53 ( Brambati et al, 2023 ). POL THETA was found in this screen, in agreement with previous studies.…”

Discovery and Proof of Concept of Potent Dual Polθ/PARP Inhibitors for Efficient Treatment of Homologous Recombination-Deficient Tumors

“…In addition, as HR-deficient tumors that have acquired PARP inhibitor resistance caused by 53BP1 loss retain Polθ overexpression and dependence, Polθ inhibition thus holds considerable potential to overcome PARP inhibitor resistance. , Consistently, bioinformatic analysis also indicated that Polθ is overexpressed and have high correlations with PARP1 in various types of tumors (Figures A,B and S1). All these findings suggest that targeting Polθ particularly in combination with PARP inhibitors would be a promising strategy in HR-deficient tumor context, including some PARP inhibitor-resistant tumors. − …”

Comparison of Multiple Strategies for Precision Transgene Knock-In in Gallus gallus Genome via Microhomology-Mediated End Joining

“…Independently from NHEJ and HDR, MMEJ is an alternative DSB repair pathway known to be active during the M-early S phases when HDR is inactive [13,19]. The evidence suggests that when NHEJ or HDR is deficient, MMEJ is a robust and efficient alternative repair option [20,21]. Generally, MMEJ is utilized in short deletions of the intervening sequence, when short microhomologies exist in the two DNA ends [13].…”