Biology subjects

Kanellou, A.

Publications and source records attributed to Kanellou, A..

3 recordsLinked to original sources

bioRxiv2024

The mitotic CIP2A-TOPBP1 axis facilitates mitotic pathway choice between MiDAS and MMEJ

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 Pol{theta} 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.

molecular biology↗

bioRxiv2024

A microscopy reporter for cGAMP reveals rare cGAS activation following DNA damage, and a lack of correlation with micronuclear cGAS enrichment

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is the primary intracellular responder to pathogen DNA. Upon DNA-binding, cGAS generates cGAMP, which binds to STING, ultimately driving inflammatory signalling. Although normally silenced on self-DNA, cGAS can be activated during genotoxic stress. A universal by-product of these conditions are micronuclei, which accumulate cGAS, and which are therefore thought to be major cGAS activators. However, due to the inability to visualise cGAS activation in single cells, this hypothesis remains largely untested. Here we solve this question with an improved intracellular cGAMP reporter, which is compatible with microscopy, flow-cytometry and plate reader setups. Surprisingly, cGAS activation in response to multiple types of genotoxic stress is limited to a subfraction of cells and does not correlate with cGAS enrichment in micronuclei. Overall, our findings suggest a revised model of innate immune signalling in response to genotoxic stress, and introduce a novel and flexible tool with which to examine this model in future.

cell biology↗

bioRxiv2023

Actin nucleators safeguard replication forks by limiting nascent strand degradation

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e., Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that {beta}-actin interacts with RPA directly in vitro, and in vivo a hyper-depolymerizing {beta}-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing {beta}-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity.

molecular biology↗