The Last Chance Saloon

Accurate chromosome segregation requires the removal of all chromatin bridges, which link chromosomes before cell division. When chromatin bridges fail to be removed, cell cycle progression may halt, or cytokinesis failure and ensuing polyploidization may occur. Conversely, the inappropriate severing of chromatin bridges leads to chromosome fragmentation, excessive genome instability at breakpoints, micronucleus formation, and chromothripsis. In this mini-review, we first describe the origins of chromatin bridges, the toxic processing of chromatin bridges by mechanical force, and the TREX1 exonuclease. We then focus on the abscission checkpoint (NoCut) which can confer a transient delay in cytokinesis progression to facilitate bridge resolution. Finally, we describe a recently identified mechanism uncovered in C. elegans where the conserved midbody associated endonuclease LEM-3/ANKLE1 is able to resolve chromatin bridges generated by various perturbations of DNA metabolism at the final stage of cell division. We also discuss how LEM-3 dependent chromatin bridge resolution may be coordinated with abscission checkpoint (NoCut) to achieve an error-free cleavage, therefore acting as a “last chance saloon” to facilitate genome integrity and organismal survival.

Catalase inhibition by nitric oxide potentiates hydrogen peroxide to trigger catastrophic chromosome fragmentation in Escherichia coli

Hydrogen peroxide (H2O2, HP) is a universal toxin that organisms deploy to kill competing or invading cells. Bactericidal action of H2O2 presents several questions. First, the lethal H2O2 concentrations in bacterial cultures are 1000x higher than, for example, those calculated for the phagosome. Second, H2O2-alone kills bacteria in cultures either by mode-one, via iron-mediated chromosomal damage, or by mode-two, via unknown targets, but the killing mode in phagosomes is unclear. Third, phagosomal H2O2 toxicity is enhanced by production of nitric oxide (NO), but in vitro studies disagree: some show NO synergy with H2O2 antimicrobial action, others instead report alleviation. To investigate this “NO paradox,” we treated Escherichia coli with various concentrations of H2O2-alone or H2O2+NO, measuring survival and chromosome stability. We found that all NO concentrations make sublethal H2O2 treatments highly lethal, via triggering catastrophic chromosome fragmentation (mode-one killing). Yet, NO-alone is not lethal, potentiating H2O2 toxicity by blocking H2O2 scavenging in cultures. Catalases represent obvious targets of NO inhibition, and catalase-deficient mutants are indeed killed equally by H2O2-alone or H2O2+NO treatments, also showing similar levels of chromosome fragmentation. Interestingly, iron chelation blocks chromosome fragmentation in catalase-deficient mutants without blocking H2O2-alone lethality, indicating mode-two killing. In fact, mode-two killing of WT cells by much higher H2O2 concentrations is transiently alleviated by NO, reproducing the “NO paradox.” We conclude that NO potentiates H2O2 toxicity by promoting mode-one killing (via catastrophic chromosome fragmentation) by otherwise static low H2O2 concentrations, while transiently suppressing mode-two killing by immediately lethal high H2O2 concentrations.

Functional characterization of Caenorhabditis elegans cbs-2 gene during meiosis

Cystathionine β-synthase (CBS) is a eukaryotic enzyme that maintains the cellular homocysteine homeostasis and catalyzes the conversion of homocysteine to L-cystathionine and Hydrogen sulfide, via the trans-sulfuration pathway. In Caenorhabditis elegans, two cbs genes are present: cbs-1 functions similarly as to human CBS, and cbs-2, whose roles are instead unknown. In the present study we performed a phenotypic characterization of the cbs-2 mutant. The null cbs-2 mutant is viable, fertile and shows the wild-type complement of six bivalents in most oocyte nuclei, which is indicative of a correct formation of crossover recombination. In absence of synaptonemal complex formation (syp-2 mutant), loss of cbs-2 leads to chromosome fragmentation, suggesting that cbs-2 is essential during inter-sister repair. Interestingly, although proficient in the activation of the DNA damage checkpoint after exposure to genotoxic stress, the cbs-2 mutant is defective in DNA damage-induced apoptosis in meiotic germ cells. These results suggest possible functions for CBS-2 in meiosis, distinct from a role in the trans-sulfuration pathway. We propose that the C. elegans CBS-2 protein is required for both inter-sister repair and execution of DNA damage-induced apoptosis.

Programmed genome rearrangements in ciliates

Ciliates are a highly divergent group of unicellular eukaryotes with separate somatic and germline genomes found in distinct dimorphic nuclei. This characteristic feature is tightly linked to extremely laborious developmentally regulated genome rearrangements in the development of a new somatic genome/nuclei following sex. The transformation from germline to soma genome involves massive DNA elimination mediated by non-coding RNAs, chromosome fragmentation, as well as DNA amplification. In this review, we discuss the similarities and differences in the genome reorganization processes of the model ciliates Paramecium and Tetrahymena (class Oligohymenophorea), and the distantly related Euplotes, Stylonychia, and Oxytricha (class Spirotrichea).

ER-directed TREX1 limits cGAS recognition of micronuclei

ABSTRACTChromosomal instability in cancer results in the formation of nuclear aberrations termed micronuclei. Spontaneous loss of micronuclear envelope integrity exposes DNA to the cytoplasm, leading to chromosome fragmentation and innate immune activation. Despite connections to cancer genome evolution and anti-tumor immunity, the mechanisms underlying damage and immune sensing of micronuclear DNA are poorly understood. Here, we use a novel method for the purification of micronuclei and live-cell imaging to show that the ER-associated nuclease TREX1 inhibits cGAS sensing of micronuclei by stably associating with and degrading micronuclear DNA upon micronuclear envelope rupture. We identify a TREX1 mutation, previously associated with autoimmune disease, that untethers TREX1 from the ER, disrupts TREX1 localization to micronuclei, alleviates micronuclear DNA damage, and enhances cGAS recognition of micronuclei. Together, these results establish ER-directed resection of micronuclear DNA by TREX1 as a critical regulator of cytosolic DNA sensing in chromosomally unstable cells and provide a mechanistic basis for the importance of TREX1 ER-tethering in preventing autoimmunity.

Reactive Oxygen Species Production Is a Major Factor Directing the Postantibiotic Effect of Fluoroquinolones in Streptococcus pneumoniae

ABSTRACT We studied the molecular mechanisms involved in the postantibiotic effect of the fluoroquinolones levofloxacin and moxifloxacin in Streptococcus pneumoniae. Wild-type strain R6 had postantibiotic effects of 2.05 ± 0.10 h (mean ± standard deviation [SD]) and 3.23 ± 0.45 h at 2.5× and 10× MIC of levofloxacin, respectively. Moxifloxacin exhibited lower effects of 0.87 ± 0.1 and 2.41 ± 0.29 h at 2.5× and 10× MIC, respectively. Fluoroquinolone-induced chromosome fragmentation was measured at equivalent postantibiotic effects for levofloxacin (2.5× MIC) and moxifloxacin (10× MIC). After 2 h of drug removal, reductions were approximately 7-fold for levofloxacin and 3-fold for moxifloxacin, without further decreases at later times. Variations in reactive oxygen species production were detected after 4 to 6 h of drug withdrawals, with decreases ≥400-fold for levofloxacin and ≥800-fold for moxifloxacin at 6 h. In accordance, after 4 to 6 h of drug withdrawal, the levofloxacin-induced upregulation of the fatCDEB operon, introducing iron in the bacteria, decreased up to 2- to 3-fold, and the moxifloxacin-induced upregulation of several genes involved in the production of pyruvate was reduced 3- to 7-fold. In accordance, lower postantibiotic effects (up to 1 h) were observed in strain R6 ΔspxB, lacking the main enzyme involved in oxygen peroxide production, than in R6. Although no change in the recovery of chromosome fragmentation was observed between R6 and R6 ΔspxB, 3.5 × 103-fold lower reactive oxygen species production was observed in R6 ΔspxB, without changes after drug removal. These results show that reactive oxygen species are the main factors directing the postantibiotic effect of levofloxacin and moxifloxacin in S. pneumoniae.

A Single Defined Sister Chromatid Fusion Destabilizes Cell Cycle through Micronuclei Formation

Chromosome fusion is deleterious among oncogenic chromosome rearrangements, and has been proposed to cause multiple tumor-driving abnormalities. Conventional methodologies, however, lack the strictness of the experimental controls on the fusion such as the exact timing, the number and the types of fusion in a given cell. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. Fused chromosome developed numerical and structural abnormalities, including chromosome fragmentation, an indicative of eventual chromothripsis. Live cell imaging and hierarchical Bayesian modeling indicated that micronucleus (MN) is generated in the first few cell cycle, and that cells with MN tend to possess cell cycle abnormalities. These results demonstrate that, although most cells can tolerate a single fusion, even a single sister chromatid fusion destabilizes cell cycle through MN formation.