Stalled replication fork protection limits cGAS–STING and P-body-dependent innate immune signalling

Emam, Ahmed A.; Wu, Xiao Y.; Xu, Shengfeng; Wang, Longqiang; Liu, Shichang; Wang, Bin

doi:10.1038/s41556-022-00950-8

Cited by 34 publications

(23 citation statements)

References 57 publications

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“…Recently, FANCD2 inhibition of DNA2 resection, surprisingly, has been demonstrated to play a significant role in restraining accumulation of cytoplasmic DNA and induction of the cGAS-STING pathway in response to replication fork stalling in vivo (Emam et al, 2022). Possible roles for FANCD2 in immune secretion and inflammation and their emerging link to DNA repair pathways lend further significance to understanding the mechanism by which FANCD2 controls DNA2.…”

Section: Discussionmentioning

confidence: 99%

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FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability

Liu

Roubal

Polaczek

et al. 2021

Preprint

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SummaryFANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea induced stalled forks. The mechanisms of the fork protection are not well studied. Here, we purified FANCD2 to study how FANCD2 regulates DNA resection at stalled forks. In vitro, we showed that FANCD2 inhibits fork degradation in two ways: 1) it inhibits DNA2 nuclease activity by directly binding to DNA2. 2) independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit various nucleases, including DNA2. More unexpectedly, FANCD2 acts as a RAD51 mediator to stimulate the strand exchange activity of RAD51, and does so by enhancing ssDNA binding of RAD51. Our work biochemically explains mechanisms by which FANCD2 protects stalled forks and further provides a simple molecular explanation for genetic interactions between FANCD2 and the BRCA2 mediator.

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Section: Discussionmentioning

confidence: 99%

Section: Protecting Stalled Forks From Degradationmentioning

confidence: 99%

FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability

Liu

Roubal

Polaczek

et al. 2021

Preprint

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“…The purity of DNA was analyzed with UV absorbance on a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, MA, USA), the DNA with OD 260/280 ratios of 1.8 to 2.0 was prepared for further quantification. A Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and a dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA) were used for DNA quantification [ 24 , 25 ]. The samples were kept at −20 °C.…”

Section: Methodsmentioning

confidence: 99%

Quantitative PCR Assays for the Strain-Specific Identification and Enumeration of Probiotic Strain Lacticaseibacillus rhamnosus X253

Zhao

Zhang²,

Liu

et al. 2022

Foods

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Probiotics are universally recognized for their health benefits, despite the fact that their effects depend on the strain. Identification and enumeration of probiotic strains are required prior to evaluating their effectiveness. Lacticaseibacillus rhamnosus X253 is a potential probiotic strain with antioxidant capacity. Comparative genomics and single nucleotide polymorphisms (SNPs) were used to identify a strain-specific locus within the holA gene for strain X253 that was distinct in 30 different L. rhamnosus strains. Using quantitative PCR, the primers and probe designed for the locus were able to distinguish L. rhamnosus X253 from the other 20 probiotic strains. The chosen locus remained stable over 19 generations. The sensitivity of the assay was 0.2 pg genomic DNA of L. rhamnosus X253, or 103 cfu/mL bacteria of this strain. In terms of repeatability and reproducibility, relative standard deviations (RSD) were less than 1% and 3%, respectively. Additionally, this assay achieved accurate enumerations of L. rhamnosus X253 in spiked milk and complex powder samples. The strain-specific assay could be used for quality control and compliance assessment of dairy products.

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“…Since cGAS is a DNA sensor which preferentially binds to double-stranded DNA [ 34 ], we wondered how ssDNA activated STING pathway. Ahmed Emam et al found that the increased cytosolic ssDNA contains ribosomal DNA that can bind to cGAS and activate of the innate immune response [ 35 ]. Kiwon Park et al found that SAMHD1 prevents R-loop formation to preserve genome integrity [ 36 ].…”

Section: Discussionmentioning

confidence: 99%

SAMHD1 silencing cooperates with radiotherapy to enhance anti-tumor immunity through IFI16-STING pathway in lung adenocarcinoma

Gao

Jiang

et al. 2022

J Transl Med

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Background Sterile alpha motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1) is a DNA end resection factor, which is involved in DNA damage repair and innate immunity. However, the role of SAMHD1 in anti-tumor immunity is still unknown. This study investigated the effects of SAMHD1 on stimulator of interferon genes (STING)-type I interferon (IFN) pathway and radiation-induced immune responses. Methods The roles of SAMHD1 in the activation of cytosolic DNA sensing STING pathway in lung adenocarcinoma (LUAD) cells were investigated with flow cytometry, immunofluorescence, immunoblotting and qPCR. The combined effects of SAMHD1 silencing and radiation on tumor cell growth and STING pathway activation were also evaluated with colony formation and CCK8 assay. The Lewis lung cancer mouse model was used to evaluate the combined efficiency of SAMHD1 silencing and radiotherapy in vivo. Macrophage M1 polarization and cytotoxic T cell infiltration were evaluated with flow cytometry. Results The single-stranded DNA (ssDNA) accumulated in the cytosol of SAMHD1-deficient lung adenocarcinoma (LUAD) cells, accompanied by upregulated DNA sensor IFN-γ-inducible protein 16 (IFI16) and activated STING pathway. The translocation of IFI16 from nucleus to cytosol was detected in SAMHD1-deficient cells. IFI16 and STING were acquired in the activation of STING-IFN-I pathway in SAMHD1-deficient cells. SAMHD1 silencing in LUAD cells promoted macrophage M1 polarization in vitro. SAMHD1 silencing synergized with radiation to activate ssDNA-STING-IFN-I pathway, inhibit proliferation, promote apoptosis and regulate cell cycle. SAMHD1 silencing cooperated with radiotherapy to inhibit tumor growth and increase CD86+MHC-IIhigh M1 proportion and CD8+ T cell infiltration in vivo. Conclusions SAMHD1 deficiency induced IFN-I production through cytosolic IFI16-STING pathway in LUAD cells. Moreover, SAMHD1 downregulation and radiation cooperated to inhibit tumor growth and enhance anti-tumor immune responses through macrophage M1 polarization and CD8+ T cell infiltration. Combination of SAMHD1 inhibition and radiotherapy may be a potentially therapeutic strategy for LUAD patients.

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Stalled replication fork protection limits cGAS–STING and P-body-dependent innate immune signalling

Cited by 34 publications

References 57 publications

FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability

FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability

Quantitative PCR Assays for the Strain-Specific Identification and Enumeration of Probiotic Strain Lacticaseibacillus rhamnosus X253

SAMHD1 silencing cooperates with radiotherapy to enhance anti-tumor immunity through IFI16-STING pathway in lung adenocarcinoma

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