Micronuclei induced by radiation, replication stress, or chromosome segregation errors do not activate cGAS-STING.

The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral infection and inhibition of autoimmunity. Recent studies have suggested that micronuclei formed by genotoxic stress can activate innate immune signaling via the cGAS-STING pathway. Here, we investigated cGAS localization, activation, and downstream signaling from micronuclei induced by ionizing radiation, replication stress, and chromosome segregation errors. Although cGAS localized to ruptured micronuclei via binding to self-DNA, we failed to observe cGAS activation; cGAMP production; downstream phosphorylation of STING, TBK1, or IRF3; nuclear accumulation of IRF3; or expression of interferon-stimulated genes. Failure to activate the cGAS-STING pathway was observed across primary and immortalized cell lines, which retained the ability to activate the cGAS-STING pathway in response to dsDNA or modified vaccinia virus infection. We provide evidence that micronuclei formed by genotoxic insults contain histone-bound self-DNA, which we show is inhibitory to cGAS activation in cells.

HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51.

DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3' to 5' polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2-4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD.

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.

Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability.

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability.

Plk1 regulates contraction of postmitotic smooth muscle cells and is required for vascular homeostasis.

Polo-like kinase 1 (PLK1), an essential regulator of cell division, is currently undergoing clinical evaluation as a target for cancer therapy. We report an unexpected function of Plk1 in sustaining cardiovascular homeostasis. Plk1 haploinsufficiency in mice did not induce obvious cell proliferation defects but did result in arterial structural alterations, which frequently led to aortic rupture and death. Specific ablation of Plk1 in vascular smooth muscle cells (VSMCs) led to reduced arterial elasticity, hypotension, and an impaired arterial response to angiotensin II in vivo. Mechanistically, we found that Plk1 regulated angiotensin II-dependent activation of RhoA and actomyosin dynamics in VSMCs in a mitosis-independent manner. This regulation depended on Plk1 kinase activity, and the administration of small-molecule Plk1 inhibitors to angiotensin II-treated mice led to reduced arterial fitness and an elevated risk of aneurysm and aortic rupture. We thus conclude that a partial reduction of Plk1 activity that does not block cell division can nevertheless impair aortic homeostasis. Our findings have potentially important implications for current approaches aimed at PLK1 inhibition for cancer therapy.

A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling.

Central to homologous recombination in eukaryotes is the RAD51 recombinase, which forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) and catalyzes strand invasion with homologous duplex DNA. Various regulatory proteins assist this reaction including the RAD51 paralogs. We recently discovered that a RAD51 paralog complex from C. elegans, RFS-1/RIP-1, functions predominantly downstream of filament assembly by binding and remodeling RAD-51-ssDNA filaments to a conformation more proficient for strand exchange. Here, we demonstrate that RFS-1/RIP-1 acts by shutting down RAD-51 dissociation from ssDNA. Using stopped-flow experiments, we show that RFS-1/RIP-1 confers this dramatic stabilization by capping the 5' end of RAD-51-ssDNA filaments. Filament end capping propagates a stabilizing effect with a 5'→3' polarity approximately 40 nucleotides along individual filaments. Finally, we discover that filament capping and stabilization are dependent on nucleotide binding, but not hydrolysis by RFS-1/RIP-1. These data define the mechanism of RAD51 filament remodeling by RAD51 paralogs.

Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells.

The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt their migration mode to their environment. While the molecular pathways involved in this transition are well documented, the role of the microenvironment in the MAT is still poorly understood. Here, we investigated how confinement and adhesion affect this transition. We report that, in the absence of focal adhesions and under conditions of confinement, mesenchymal cells can spontaneously switch to a fast amoeboid migration phenotype. We identified two main types of fast migration--one involving a local protrusion and a second involving a myosin-II-dependent mechanical instability of the cell cortex that leads to a global cortical flow. Interestingly, transformed cells are more prone to adopt this fast migration mode. Finally, we propose a generic model that explains migration transitions and predicts a phase diagram of migration phenotypes based on three main control parameters: confinement, adhesion, and contractility.

Centralspindlin links the mitotic spindle to the plasma membrane during cytokinesis.

At the end of cell division, cytokinesis splits the cytoplasm of nascent daughter cells and partitions segregated sister genomes. To coordinate cell division with chromosome segregation, the mitotic spindle controls cytokinetic events at the cell envelope. The spindle midzone stimulates the actomyosin-driven contraction of the cleavage furrow, which proceeds until the formation of a microtubule-rich intercellular bridge with the midbody at its centre. The midbody directs the final membrane abscission reaction and has been proposed to attach the cleavage furrow to the intercellular bridge. How the mitotic spindle is connected to the plasma membrane during cytokinesis is not understood. Here we identify a plasma membrane tethering activity in the centralspindlin protein complex, a conserved component of the spindle midzone and midbody. We demonstrate that the C1 domain of the centralspindlin subunit MgcRacGAP associates with the plasma membrane by interacting with polyanionic phosphoinositide lipids. Using X-ray crystallography we determine the structure of this atypical C1 domain. Mutations in the hydrophobic cap and in basic residues of the C1 domain of MgcRacGAP prevent association of the protein with the plasma membrane, and abrogate cytokinesis in human and chicken cells. Artificial membrane tethering of centralspindlin restores cell division in the absence of the C1 domain of MgcRacGAP. Although C1 domain function is dispensable for the formation of the midzone and midbody, it promotes contractility and is required for the attachment of the plasma membrane to the midbody, a long-postulated function of this organelle. Our analysis suggests that centralspindlin links the mitotic spindle to the plasma membrane to secure the final cut during cytokinesis in animal cells.

Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C.

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.

Targeting of the RhoGEF Ect2 to the equatorial membrane controls cleavage furrow formation during cytokinesis.

In animal cells, formation of the cytokinetic furrow requires activation of the GTPase RhoA by the guanine nucleotide exchange factor Ect2. How Ect2, which is associated with the spindle midzone, controls RhoA activity at the equatorial cortex during anaphase is not understood. Here, we show that Ect2 concentrates at the equatorial membrane during cytokinesis in live cells. Ect2 membrane association requires a pleckstrin homology domain and a polybasic cluster that bind to phosphoinositide lipids. Both guanine nucleotide exchange function and membrane targeting of Ect2 are essential for RhoA activation and cleavage furrow formation in human cells. Membrane localization of Ect2 is spatially confined to the equator by centralspindlin, Ect2's spindle midzone anchor complex, and is temporally coordinated with chromosome segregation through the activation state of CDK1. We propose that targeting of Ect2 to the equatorial membrane represents a key step in the delivery of the cytokinetic signal to the cortex.

Rapid exchange of bound ADP on the Staphylococcus aureus replication initiation protein DnaA.

In Escherichia coli, regulatory inactivation of the replication initiator DnaA occurs after initiation as a result of hydrolysis of bound ATP to ADP, but it has been unknown how DnaA is controlled to coordinate cell growth and chromosomal replication in gram-positive bacteria such as Staphylococcus aureus. This study examined the roles of ATP binding and its hydrolysis in the regulation of the S. aureus DnaA activity. In vitro, S. aureus DnaA melted S. aureus oriC in the presence of ATP but not ADP by a mechanism independent of ATP hydrolysis. Unlike E. coli DnaA, binding of ADP to S. aureus DnaA was unstable. As a result, at physiological concentrations of ATP, ADP bound to S. aureus DnaA was rapidly exchanged for ATP, thereby regenerating the ability of DnaA to form the open complex in vitro. Therefore, we examined whether formation of ADP-DnaA participates in suppression of replication initiation in vivo. Induction of the R318H mutant of the AAA+ sensor 2 protein, which has decreased intrinsic ATPase activity, caused over-initiation of chromosome replication in S. aureus, suggesting that formation of ADP-DnaA suppresses the initiation step in S. aureus. Together with the biochemical features of S. aureus DnaA, the weak ability to convert ATP-DnaA into ADP-DnaA and the instability of ADP-DnaA, these results suggest that there may be unidentified system(s) for reducing the cellular ratio of ATP-DnaA to ADP-DnaA in S. aureus and thereby delaying the re-initiation of DNA replication.

Polo-like kinase 1 directs assembly of the HsCyk-4 RhoGAP/Ect2 RhoGEF complex to initiate cleavage furrow formation.

To complete cell division with high fidelity, cytokinesis must be coordinated with chromosome segregation. Mammalian Polo-like kinase 1, Plk1, may function as a critical link because it is required for chromosome segregation and establishment of the cleavage plane following anaphase onset. A central spindle-localized pool of the RhoGEF Ect2 promotes activation of the small GTPase RhoA, which drives contractile ring assembly at the equatorial cortex. Here, we have investigated how Plk1 promotes the central spindle recruitment of Ect2. Plk1 phosphorylates the noncatalytic N terminus of the RhoGAP HsCyk-4 at the central spindle, creating a phospho-epitope recognized by the BRCA1 C-terminal (BRCT) repeats of Ect2. Failure to phosphorylate HsCyk-4 blocks Ect2 recruitment to the central spindle and the subsequent induction of furrowing. Microtubules, as well as the microtubule-associated protein (MAP) Prc1, facilitate Plk1 phosphorylation of HsCyk-4. Characterization of a phosphomimetic version of HsCyk-4 indicates that Plk1 promotes Ect2 recruitment through multiple targets. Collectively, our data reveal that formation of the HsCyk-4-Ect2 complex is subject to multiple layers of regulation to ensure that RhoA activation occurs between the segregated sister chromatids during anaphase.

Polo-like kinase 1 reaches beyond mitosis--cytokinesis, DNA damage response, and development.

Polo-like kinase 1 (Plk1) is a key regulator of cell division in eukaryotic cells. In this review we focus on recent leaps in our understanding of how Plk1 controls cytokinesis, the final stage of cell division. Furthermore, we will go beyond mitosis to highlight unexpected roles of Plk1 during interphase and during animal development. In vertebrate cells, Plk1 has emerged as a novel player in maintaining genomic stability during DNA replication and as an important modulator of the DNA damage checkpoint. Plk1 functions extend past the 'core' cell cycle. Plk1 acts as a link between developmental processes and the cell cycle machinery during asymmetric cell divisions in flies and worms. The term 'mitotic kinase' might not do justice to Plk1 in the light of these recent results.

Preferences for phosphorylation sites in the retinoblastoma protein of D-type cyclin-dependent kinases, Cdk4 and Cdk6, in vitro.

D-type cyclin-dependent kinases (Cdk4 and Cdk6) regulate the G1 to S phase progression of the mammalian cell cycle. It has been suggested that Cdk4 and Cdk6 may have distinct functions in vivo, even though they are indistinguishable biochemically. Here we show that although these Cdks phosphorylate multiple residues in pRB, they do so with different residue selectivities in vitro; Thr821 and Thr826 are preferentially phosphorylated by Cdk6 and Cdk4, respectively. This raises the possibility different substrate specificities lead to their different roles in the regulation of cellular events. Furthermore, our results indicate the new concept that Cdk itself contributes to substrate recognition.

Cdk-mediated phosphorylation of pRB regulates HDAC binding in vitro.

Retinoblastoma protein (pRB) controls the G1/S transition in the cell cycle by binding and inactivating E2F transcription factor. pRB changes the chromatin structure at the E2F-responsive promoter by recruiting histone deacetylase (HDAC) to the pRB-E2F complex, thus controlling the transcriptional activity of E2F. Cyclin-dependent kinases (Cdks) phosphorylate pRB and disrupt association between pRB and E2F. We investigated the effects of pRB phosphorylation on HDAC-1 binding in vitro. Phosphorylation of pRB by Cdk4-cyclin D2, Cdk2-cyclin E, and Cdk2-cyclin A inhibited association of pRB with HDAC. Among these Cdks, Cdk4-cyclin D2 showed particularly effective inhibition of pRB-HDAC complex formation. Using pRB mutants with various deletions in the N- and C-terminal domains, we found that both the pocket and C-terminal domains are important for regulating association between pRB and HDAC.

ICRF-193, a catalytic inhibitor of DNA topoisomerase II, inhibits re-entry into the cell division cycle from quiescent state in mammalian cells.

BACKGROUND: To describe the requirement of DNA topoisomerase II (topo II) during transition from the quiescent state (G0 phase) to the cell division cycle in mammalian cells, we examined the influence of ICRF-193, a catalytic inhibitor of topo II, on re-entry into the cell division cycle of quiescent cells in response to appropriate growth stimuli. RESULTS: The re-entry into the S phase of cultured cell lines arrested at the quiescent (G0) phase by serum-starvation was sensitive to 10 microm ICRF-193. DNA syntheses induced by lipopolysaccharide in murine spleen cells or by release from contact-inhibition were also inhibited by ICRF-193. The cell lines with a high-level of resistance toward ICRF-193 due to a point mutation in the topo IIalpha gene entered into the S phase from quiescence in the presence of ICRF-193. The drug did not inhibit entry into the S phase in cultured cells released from arrest at the metaphase or G1 phase. CONCLUSION: There is an ICRF-193-sensitive step during re-entry of quiescent mammalian cells into the cell division cycle upon growth stimulation and the drug targets topo IIalpha during the process.