Antony W. Oliver

Most Triple Negative Breast Cancers (TNBCs) are p53 mutant high-grade invasive ductal carcinomas with a basal-like transcriptional programme. A large proportion of TNBCs also have mutation signatures indicating defective homologous recombination (HR) DNA repair. While HR deficiency is mutagenic and oncogenic, it can also compromise cell fitness. Here, we show that BLBCs harbouring the recurrent 9p21.3–9p24 amplicon, and particularly those that are HR-defective, overexpress PUM3 (Pumilio RNA binding family member 3). PUM3 inhibition is also synthetic lethal in tumour cells that overexpress PUM3 including those that have BRCA1/BRCA2 defects. Mechanistically, PUM3 suppresses transcription-mediated replicative stress and R-loop accumulation, thereby preventing DNA damage that would normally require RAD51-mediated repair. Transcription/replication collisions are commonly resolved by topoisomerase TOP1; without PUM3, TOP1 DNA chromatin localisation is impaired, revealing one mechanism by which PUM3 limits replication stress. PUM3 is therefore required for the continued fitness of a BLBC subset, allowing tolerance of HR deficiency’s deleterious effects.

The BTRR (BLM/TOP3A/RMI1/RMI2) complex resolves DNA replication and recombination intermediates, as well as mitotic DNA intertwinements, yet how its diverse activities are regulated remains unclear. Here, we identify key determinants of stable assembly of the BTRR complex on ultrafine DNA bridges (UFBs). We reveal that the RMI1-RMI2 subcomplex acts as a molecular reader of hydrophobic-rich short linear motifs (SLiMs) embedded within PICH and FANCM, thereby providing a conserved mechanism for engaging distinct Superfamily2 DNA translocases during chromosome segregation and DNA repair. During UFB resolution, SLiM binding stabilises the PICH-BTRR complex assembly and supports essential interactions among BLM, PICH, and DNA. Persistent PICH-BTRR complex association post-mitosis would compromise interphase DNA damage responses and is prevented by active PICH nuclear exclusion. Our findings define the molecular basis of UFB-binding complex assembly and highlight its spatiotemporal control required to specialise BTRR complex functions.

SUMMARY: EIF2α kinase heme-regulated inhibitor (HRI) is a novel target for haematological malignancies with modulators reported to trigger cell death via the HRI-eIF2α-ATF4 pathway. We report a protocol for producing the minimal kinase domain of full-length human HRI, termed ‘HRI _KD-ΔKI ,’ where the unstructured 140 amino acid (aa) kinase insert (KI) within HRI kinase domain (HRI _KD ) is replaced with a 2aa glycine/serine (GS) linker. X-ray crystal structures were determined of ‘apo’-HRI _KD-ΔKI and of its complex with ATP at 2.1 & 2.5 Å resolution respectively. Both structures display a canonical bi-lobal kinase fold. However, they remain in a non-productive state with a displaced C-helix, disassembled R-spine, and a disordered activation segment hindering the substrate site. Biophysical assays (fluorescence based thermal shift & Synchrotron Radiation Circular Dichroism) demonstrate HRI _KD-ΔKI retains its functional ligand-binding conformation. All together, these findings define structural and ligand-binding features of HRI to support ongoing drug discovery efforts in blood cancer.

Break-induced replication (BIR) facilitates single-ended DNA double-strand break (DSB) repair. Upon homologous recombination-mediated strand invasion into a homologous repair template, BIR is catalysed by a minimal replisome comprising PCNA, DNA polymerase δ (Pol δ), and the Pif1 helicase. Here, we identify an interaction between Pol δ and single-stranded DNA (ssDNA)-binding protein RPA mediated by an RPA-binding module (RBM) within Pol δ subunit Pol32 and RPA subunit Rfa1. Pol32 RBM phosphorylation at Thr256 and Thr257 increases its affinity for Rfa1, while corresponding phospho-mimetic amino-acid substitutions promote BIR efficiency in vivo. This suggests that Pol32 functions as a rheostat whose phosphorylation enhances Pol δ’s affinity for RPA-bound BIR intermediates, thereby boosting BIR efficiency. Modelling indicates that Pol32 phospho-RBM-Rfa1 interactions mirror the binding mode of RBMs in Pif1 and the FANCM helicase and BIR antagonist Mph1. This implies a key role for RPA in the dynamic orchestration of the enzymes mediating BIR.

Mitotic DNA double-strand breaks (DSBs) accumulate in response to replication stress or BRCA1/2 deficiency posing a significant threat to genome stability as repair by non-homologous end-joining (NHEJ) and homologous recombination (HR) is largely inactivated in mitosis. Instead, mitotic cells rely on alternative repair processes such as microhomology-mediated end-joining (MMEJ) and mitotic DNA synthesis (MiDAS). How these mitotic DNA repair pathways are functionally regulated remains unclear. Here we reveal that the CIP2A-TOPBP1 complex plays an essential regulatory role by facilitating the mitotic recruitment of both SMX complex components and Polθ to mitotic chromatin. Recruitment of the SMX complex components is driven by CDK1-dependent phosphorylation of SLX4 at Thr1260, enabling its interaction with TOPBP1 BRCT domains 1/2, thereby promoting MiDAS. Concurrently, CIP2A promotes efficient mitotic localisation of Polθ to facilitate MMEJ. The simultaneous functional disruption of both MiDAS and MMEJ pathways upon CIP2A loss provides rationale for the synthetic lethality observed in BRCA1 or 2-deficient cells. These findings position the CIP2A-TOPBP1 axis as a central regulatory hub for mitotic DNA repair, highlighting therapeutic opportunities in tumours characterised by HR deficiency or elevated replication stress.

SUMMARY: DNA double-strand breaks and unresolved DNA replication intermediates are particularly dangerous during mitosis. Paradoxically, cells inactivate canonical DNA repair mechanisms during chromosome segregation in favor of alternative pathways that depend on TOPBP1 and CIP2A, but how they function is still poorly defined. Here, we describe the identification of DDIAS as a mitosis-specific DNA damage response protein. We establish DDIAS as a phosphorylation-dependent component and effector of the TOPBP1-CIP2A complex with single-stranded DNA (ssDNA)-binding activity, and thereby delineate a ssDNA protection mechanism that safeguards chromosome integrity during mitosis, particularly in BRCA-deficient cells. We also identify biallelic inactivating mutations in DDIAS and CIP2A in patients with severe neurodevelopmental phenotypes. These findings highlight the physiological importance of the DNA damage response in mitosis, and may open new avenues for synthetic-lethal therapy in BRCA-deficient cancers.

The Greatwall kinase inhibits PP2A-B55 phosphatase activity during mitosis to stabilise critical Cdk1-driven mitotic phosphorylation. Although Greatwall represents a potential oncogene and prospective therapeutic target, our understanding of the cellular and molecular consequences of chemical Greatwall inactivation remains limited. To address this, we introduce C-604, a highly selective Greatwall inhibitor, and characterise both immediate and long-term cellular responses to the chemical attenuation of Greatwall activity. We demonstrate that Greatwall inhibition causes systemic destabilisation of the mitotic phosphoproteome, premature mitotic exit and pleiotropic cellular pathologies. Importantly, we show that the cellular and molecular abnormalities associated with reduced Greatwall activity are specifically dependent on the B55α isoform, rather than other B55 variants, underscoring PP2A-B55α phosphatases as key mediators of the cytotoxic effects of Greatwall-targeting agents in human cells. Additionally, we establish that sensitivity to Greatwall inhibition varies in different cell line models and that dependency on Greatwall activity reflects the balance between Greatwall and B55α expression levels. Our findings highlight Greatwall dependency as a cell-specific vulnerability and propose the B55α-to-Greatwall expression ratio as a predictive biomarker of cellular responses to Greatwall-targeted therapeutics.

The BTRR (BLM/TOP3A/RMI1/RMI2) complex resolves DNA replication and recombination intermediates to maintain genome stability. Alongside PICH, they target mitotic DNA intertwinements, known as ultrafine DNA bridges, facilitating chromosome segregation. Both BLM and PICH undergo transient mitotic hyper-phosphorylation, but the biological significance of this remains elusive. Here, we uncover that during early mitosis, CDK1 and PLK1 constrain BTRR complex activities at centromeres. CDK1 destabilises the complex, limiting its binding to PICH at specialised chromatin underneath kinetochores. Inactivating the BLM-TOP3A interaction compromises the UFB-binding complex functions and prevents centromere destruction. Different phosphorylation on BLM affects the TRR subcomplex interaction and the mitotic activity, particularly phosphorylation at Ser144 and multiple PLK1-target sites suppresses illegitimate centromeric DNA unwinding. However, unleashing such activity after sister-chromatid cohesion inactivation facilitates the separation of entangled chromosomes. Here, we show a centromere protection pathway in human mitotic cells, heavily reliant on a tight spatiotemporal control of the BTRR complex.

RIF1 is a multifunctional protein that regulates DNA replication and repair. RIF1-deficient cells are hypersensitive to DNA replication stress. Of the two alternatively spliced RIF1 isoforms, called RIF1-Short and RIF1-Long, the RIF1-Long isoform is more capable than RIF1-Short in supporting cell recovery from replication stress. Examining replication stress resistance mechanisms specific to RIF1-Long, we find that prolonged replication stress unexpectedly induces interaction of RIF1-Long with BRCA1. Mechanistically, a phosphorylated SPKF motif unique to the RIF1-Long isoform binds the tandem BRCT domain of BRCA1. BRCA1–RIF1-Long interaction is strongly down-regulated through dephosphorylation by RIF1-associated Protein Phosphatase 1. BRCA1–RIF1-Long interaction requires ATR signaling, and occurs predominantly during S phase. Loss of RIF1-Long impairs the formation of RAD51 foci, and reduces the efficiency of homology-mediated repair at broken replication forks. In summary, our investigation establishes RIF1-Long as a new functional binding partner of the BRCA1-BRCT domain, crucial to protect cells from extended DNA replication stress by enabling RAD51-dependent repair of broken replication forks.

Mitotic DNA double-strand breaks (DSBs) accumulate in response to replication stress or BRCA1/2 deficiency posing a significant threat to genome stability as repair by non-homologous end-joining (NHEJ) and homologous recombination (HR) is inactivated in mitosis. Mitotic cells instead rely on the mechanisms of microhomology mediated end-joining (MMEJ) and mitotic DNA synthesis (MiDAS). However, how these pathways are regulated in mitosis remains unknown. Here we reveal the CIP2A-TOPBP1 complex facilitates recruitment of SMX complex components to mitotic chromatin marked by CIP2A, through a CDK1-dependent interaction between TOPBP1 BRCT 1/2 and SLX4 phospho-threonine1260, that drives MiDAS. Furthermore, CIP2A promotes the recruitment of Polθ to facilitate mitotic MMEJ. This defines a mechanistic framework for mitotic DSB repair, where simultaneous disruption of MiDAS and MMEJ pathways underpins the synthetic lethality observed in BRCA1/2-deficient cells following CIP2A depletion. These findings provide critical insights into mitotic DNA repair and highlights therapeutic opportunities in HR deficient tumours.