Sensing of cytoplasmic DNA by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) results in production of the dinucleotide cGAMP and consecutive activation of stimulator ...of interferon genes (STING) followed by production of type I interferon (IFN). Although cancer cells contain supra-normal concentrations of cytoplasmic DNA, they rarely produce type I IFN spontaneously. This suggests that defects in the DNA-sensing pathway may serve as an immune escape mechanism. We find that cancer cells produce cGAMP that is transferred via gap junctions to tumor-associated dendritic cells (DCs) and macrophages, which respond by producing type I IFN in situ. Cancer-cell-intrinsic expression of cGAS, but not STING, promotes infiltration by effector CD8+ T cells and consequently results in prolonged survival. Furthermore, cGAS-expressing cancers respond better to genotoxic treatments and immunotherapy. Thus, cancer-cell-derived cGAMP is crucial to protective anti-tumor CD8+ T cell immunity. Consequently, cancer-cell-intrinsic expression of cGAS determines tumor immunogenicity and makes tumors hot. These findings are relevant for genotoxic and immune therapies for cancer.
Display omitted
•cGAS in cancer and STING in host cells are minimal requirements to activate CD8+ T cells•Cancer cells transfer cGAMP to myeloid cells in the TME that make STING-dependent IFN-I•Cancer-cell-intrinsic cGAS improves tumor immunogenicity and response to therapy
Schadt et al. show that cancer-cell-derived cGAMP is transferred to tumor-associated myeloid cells. Here, cGAMP activates STING and induces production of type I interferon. This promotes infiltration of protective CD8+ T cells and improves survival as well as response to therapy.
Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal ...induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.
Display omitted
•R-loop-induced fork stalling is followed by MUS81-dependent replication restart•RECQ5 mediates the switch from fork stalling to restart by suppressing fork reversal•Restart of R-loop-stalled forks is mediated by fork cleavage and religation•Restart of R-loop-stalled forks requires reactivation of transcription
Transcription-replication conflicts associated with the formation of R-loops represent a major cause of replication stress. Chappidi et al. reveal that replication forks blocked by co-transcriptional R-loops can be restarted by fork cleavage and religation linked to transcription restart.
Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in ...S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate β-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of β-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage.
Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by ...exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.
R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops ...play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.
Abstract
Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double-strand breaks. Here, we show that H. pylori-induced DNA damage occurs ...co-transcriptionally in S-phase cells that activate NF-kB signaling upon innate immune recognition of LPS biosynthetic intermediates, such as ADP-heptose, by the ALPK1/TIFA pathway. DNA damage is accompanied by replication fork stalling as determined by DNA fiber assay and can be observed also in gastric organoids derived from bariatric surgery patients. We link H. pylori-induced DNA damage to the formation of RNA/DNA hybrids (R-loops), as overexpression of the R-loop-processing enzyme RNAse H1 prevents DNA damage and replication stress. R-loops form in infected cells as a consequence of ADP-heptose/ALPK1/TIFA/NF-kB signaling. Factors associated with R-loop processing and prevention are recurrently mutated in gastric cancer. In summary, our results link bacterial infection and NF-kB-driven innate immune responses to DNA damage, replication stress, and carcinogenesis.
Citation Format: Michael Bauer, Zuzana Nascakova, Anca Mihai, Anne Müller. The ALPK1/TIFA/NF-kB axis links a bacterial carcinogen to replication stress and DNA damage abstract. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B34.
R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co-transcriptionally in the opposite ...orientation to replication fork progression. A recent study has shown that replication forks stalled by co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by ELL-dependent reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds R-loops in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4-ELL pathway for resolution of R-loop-mediated transcription-replication conflicts, which may be involved in R-loop unwinding.
Helicobacter pylori
infection is a major risk factor for the development of gastric cancer. The bacteria reside in close proximity to gastric surface mucous as well as stem and progenitor cells. ...Here, we take advantage of wild-type and genetically engineered murine gastric organoids and organoid-derived monolayers to study the cellular targets of
H. pylori
–induced DNA damage and replication stress and to explore possible interactions with preexisting gastric cancer driver mutations. We find using alkaline comet assay, single-molecule DNA fiber assays, and immunofluorescence microscopy of DNA repair foci that
H. pylori
induces transcription-dependent DNA damage in actively replicating, Leucine-rich-repeat containing G-Protein-Coupled Receptor 5 (Lgr5)–positive antral stem and progenitor cells and their Troy-positive corpus counterparts, but not in other gastric epithelial lineages. Infection-dependent DNA damage is aggravated by
Apc
inactivation, but not by
Trp53
or
Smad4
loss, or
Erbb2
overexpression. Our data suggest that
H. pylori
induces DNA damage in stem and progenitor cells, especially in settings of hyperproliferation due to constitutively active Wnt signaling.
Stem cells sustain DNA damage upon exposure to
Helicobacter pylori
that is aggravated by constitutively active Wnt signaling.
One of the most common infections of a human organism is an infection of stomach induced by pathogenic bacteria Helicobacter pylori (H. pylori). It is estimated that every second person is infected, ...with even higher prevalence in developing countries. As a quiet enemy, H. pylori can colonise a human stomach for decades without manifestation of infection-associated symptoms. However, chronic infection may cause severe damage to the stomach tissue, subsequently leading to the development of gastric diseases, including gastritis and ulcer disease. H. pylori infection is also a driving cause of gastric cancer, with 80% of gastric cancers being associated with chronic infection. H. pylori ensures its life-long persistence in a human host organism via the action of its virulence factors, which have a pleiotropic effect on multiple systems, mostly acting on the attenuation of a human immune system and the induction of atrophy of stomach tissue. The irreversible changes of stomach epithelium are induced by activation of an innate immune response in H. pylori-exposed epithelial cells through the stimulation of ALPK1/TIFA/NF-κB signalling pathway upon a recognition of β-ADP heptose, an intermediate product of bacterial lipopolysaccharide biosynthesis, and consequently leading to the formation of DNA...
The paper describes the impact of factors which influence the building's energy consumption. Building constructions are important factor that influences total energy necessity for heating, where ...energetic efficiency presents way to better competition, since it decrease service costs. Building is greatest consumer of energy and here lies also potential of rationalization of energy costs. During quantification of energy savings there were used two methods: upside down - evaluation of savings through obligatory indicators in individual sectors of national economy and bottom-up - detail evaluation of individual projects. We considered with project's financing from own sources. Choice of optimal variant had been made by the way of basic variant. From the view of economical evaluation we calculated net present value, and by this way we discovered project with complex renovation of building presents optimal variant that shows better preferences. Realization of measurements can help to achieve decreasing of energy consumption, as well as costs on energy. There is immediately achieved total costs savings, and by this way payback period is shortening.
PDF
