Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) promotes stress granule formation via YBX1 phosphorylation in ovarian cancer.

APE1 is an essential gene involved in DNA damage repair, the redox regulation of transcriptional factors (TFs) and RNA processing. APE1 overexpression is common in cancers and correlates with poor patient survival. Stress granules (SGs) are phase-separated cytoplasmic assemblies that cells form in response to environmental stresses. Precise regulation of SGs is pivotal to cell survival, whereas their dysregulation is increasingly linked to diseases. Whether APE1 engages in modulating SG dynamics is worthy of investigation. In this study, we demonstrate that APE1 colocalizes with SGs and promotes their formation. Through phosphoproteome profiling, we discover that APE1 significantly alters the phosphorylation landscape of ovarian cancer cells, particularly the phosphoprofile of SG proteins. Notably, APE1 promotes the phosphorylation of Y-Box binding protein 1 (YBX1) at S174 and S176, leading to enhanced SG formation and cell survival. Moreover, expression of the phosphomutant YBX1 S174/176E mimicking hyperphosphorylation in APE1-knockdown cells recovered the impaired SG formation. These findings shed light on the functional importance of APE1 in SG regulation and highlight the importance of YBX1 phosphorylation in SG dynamics.

Characterization of a Novel N4-Methylcytosine Restriction-Modification System in Deinococcus radiodurans.

Deinococcus radiodurans is an extremophilic microorganism that possesses a unique DNA damage repair system, conferring a strong resistance to radiation, desiccation, oxidative stress, and chemical damage. Recently, we discovered that D. radiodurans possesses an N4-methylation (m4C) methyltransferase called M.DraR1, which recognizes the 5'-CCGCGG-3' sequence and methylates the second cytosine. Here, we revealed its cognate restriction endonuclease R.DraR1 and recognized that it is the only endonuclease specially for non-4C-methylated 5'-CCGCGG-3' sequence so far. We designated the particular m4C R.DraR1-M.DraR1 as the DraI R-M system. Bioinformatics searches displayed the rarity of the DraI R-M homologous system. Meanwhile, recombination and transformation efficiency experiments demonstrated the important role of the DraI R-M system in response to oxidative stress. In addition, in vitro activity experiments showed that R.DraR1 could exceptionally cleave DNA substrates with a m5C-methlated 5'-CCGCGG-3' sequence instead of its routine activity, suggesting that this particular R-M component possesses a broader substrate choice. Furthermore, an imbalance of the DraI R-M system led to cell death through regulating genes involved in the maintenance of cell survival such as genome stability, transporter, and energy production. Thus, our research revealed a novel m4C R-M system that plays key roles in maintaining cell viability and defending foreign DNA in D. radiodurans.

The Deinococcus protease PprI senses DNA damage by directly interacting with single-stranded DNA.

Bacteria have evolved various response systems to adapt to environmental stress. A protease-based derepression mechanism in response to DNA damage was characterized in Deinococcus, which is controlled by the specific cleavage of repressor DdrO by metallopeptidase PprI (also called IrrE). Despite the efforts to document the biochemical, physiological, and downstream regulation of PprI-DdrO, the upstream regulatory signal activating this system remains unclear. Here, we show that single-stranded DNA physically interacts with PprI protease, which enhances the PprI-DdrO interactions as well as the DdrO cleavage in a length-dependent manner both in vivo and in vitro. Structures of PprI, in its apo and complexed forms with single-stranded DNA, reveal two DNA-binding interfaces shaping the cleavage site. Moreover, we show that the dynamic monomer-dimer equilibrium of PprI is also important for its cleavage activity. Our data provide evidence that single-stranded DNA could serve as the signal for DNA damage sensing in the metalloprotease/repressor system in bacteria. These results also shed light on the survival and acquired drug resistance of certain bacteria under antimicrobial stress through a SOS-independent pathway.

MoaE Is Involved in Response to Oxidative Stress in Deinococcus radiodurans.

Molybdenum ions are covalently bound to molybdenum pterin (MPT) to produce molybdenum cofactor (Moco), a compound essential for the catalytic activity of molybdenum enzymes, which is involved in a variety of biological functions. MoaE is the large subunit of MPT synthase and plays a key role in Moco synthesis. Here, we investigated the function of MoaE in Deinococcus radiodurans (DrMoaE) in vitro and in vivo, demonstrating that the protein contributed to the extreme resistance of D. radiodurans. The crystal structure of DrMoaE was determined by 1.9 Å resolution. DrMoaE was shown to be a dimer and the dimerization disappeared after Arg110 had been mutated. The deletion of drmoaE resulted in sensitivity to DNA damage stress and a slower growth rate in D. radiodurans. The increase in drmoaE transcript levels the and accumulation of intracellular reactive oxygen species levels under oxidative stress suggested that it was involved in the antioxidant process in D. radiodurans. In addition, treatment with the base analog 6-hydroxyaminopurine decreased survival and increased intracellular mutation rates in drmoaE deletion mutant strains. Our results reveal that MoaE plays a role in response to external stress mainly through oxidative stress resistance mechanisms in D. radiodurans.

Mechanisms of helicase activated DNA end resection in bacteria.

DNA end resection mediated by the coordinated action of nuclease and helicase is a crucial step in initiating homologous recombination. The end-resection apparatus NurA nuclease and HerA helicase are present in both archaea and bacteria. Here, we report the cryo-electron microscopy structure of a bacterial HerA-NurA complex from Deinococcus radiodurans. The structure reveals a barrel-like hexameric HerA and a distinctive NurA dimer subcomplex, which has a unique extended N-terminal region (ENR) involved in bacterial NurA dimerization and activation. In addition to the long protruding linking loop and the C-terminal α helix of NurA, the flexible ENR is close to the HerA-NurA interface and divides the central channel of the DrNurA dimer into two halves, suggesting a possible mechanism of DNA end processing. In summary, this work provides new insights into the structure, assembly, and activation mechanisms of bacterial DNA end resection mediated by a minimal end-resection apparatus.

Structural and Functional Characterization of the Holliday Junction Resolvase RuvC from Deinococcus radiodurans.

Holliday junctions (HJs) are four-way DNA structures, which are an important intermediate in the process of homologous recombination. In most bacteria, HJs are cleaved by specific nucleases called RuvC resolvases at the end of homologous recombination. Deinococcus radiodurans is an extraordinary radiation-resistant bacterium and is known as an ideal model organism for elucidating DNA repair processes. Here, we described the biochemical properties and the crystal structure of RuvC from D. radiodurans (DrRuvC). DrRuvC exhibited an RNase H fold that belonged to the retroviral integrase family. Among many DNA substrates, DrRuvC specifically bound to HJ DNA and cleaved it. In particular, Mn2+ was the preferred bivalent metal co-factor for HJ cleavage, whereas high concentrations of Mg2+ inhibited the binding of DrRuvC to HJ. In addition, DrRuvC was crystallized and the crystals diffracted to 1.6 Å. The crystal structure of DrRuvC revealed essential amino acid sites for cleavage and binding activities, indicating that DrRuvC was a typical resolvase with a characteristic choice for metal co-factor.

Mechanism of genome instability mediated by human DNA polymerase mu misincorporation.

Pol µ is capable of performing gap-filling repair synthesis in the nonhomologous end joining (NHEJ) pathway. Together with DNA ligase, misincorporation of dGTP opposite the templating T by Pol µ results in a promutagenic T:G mispair, leading to genomic instability. Here, crystal structures and kinetics of Pol µ substituting dGTP for dATP on gapped DNA substrates containing templating T were determined and compared. Pol µ is highly mutagenic on a 2-nt gapped DNA substrate, with T:dGTP base pairing at the 3' end of the gap. Two residues (Lys438 and Gln441) interact with T:dGTP and fine tune the active site microenvironments. The in-crystal misincorporation reaction of Pol µ revealed an unexpected second dGTP in the active site, suggesting its potential mutagenic role among human X family polymerases in NHEJ.

Colonized extremophile Deinococcus radiodurans alleviates toxicity of cadmium and lead by suppressing heavy metal accumulation and improving antioxidant system in rice.

Cadmium (Cd) and lead (Pb) are the major toxic heavy metals accumulated in rice and pose a serious threat to human health. The most important remediation strategy is to reduce the translocation of these heavy metals from polluted soil to rice. Bioremediation using microorganisms had been widely used for preventing environmental heavy metal pollution, and the interaction between microorganisms and plants is critical to reduce the heavy metal stress. In this study, we demonstrated that an extremophile Deinococcus radiodurans, especially its mutant strain-Δdr2577 which is deficient in cell surface-layer, could efficiently prevent the translocation and damages of Cd or Pb in rice. The bacterial cells efficiently removed Cd or Pb from culture medium. Following colonization of Δdr2577 cells in rice root, Cd level decreased to 71.6% in root and 60.9% in shoot, comparing to the plants treated with Cd alone; Pb level decreased to 73.3% in root and 56.9% in shoot, comparing to the plants treated with Pb alone. Meanwhile, the bacterial cells released their intracellular antioxidant-related molecules including glutamate and manganese ions into culture medium. Accumulation of glutamate and manganese ions detected in rice root and shoot ameliorate Cd/Pb-induced oxidative stress as indicated by reduced levels of ROS and enhanced activities of antioxidant enzymes in rice. Our results provide a potential application of an extremophile bacterium in alleviating heavy metal toxicity in rice. The main findings of the work reveal the interaction between the D. radiodurans and rice, as well as the alleviating mechanism of Cd and Pb toxicity through suppressing heavy metal accumulation and improving the antioxidant system in rice by the extremophile bacterium.

Transmembrane protein DCBLD2 is correlated with poor prognosis and affects phenotype by regulating epithelial-mesenchymal transition in human glioblastoma cells.

OBJECTIVE: We attempt to investigate the biological function of the discoidin, complement C1r/C1s,Uegf, and Bmp1 and Limulus factor C, Coch, and Lgl domain-containing 2 (DCBLD2) in glioblastoma, as well as its effect on the epithelial-mesenchymal transition (EMT) process. METHODS: The public expression data of glioblastoma samples and normal brain samples from The Cancer Genome Atlas database, Genotype-Tissue Expression database and Chinese Glioma Genome Atlas database were used to analyze the expression of DCBLD2 and its relationship with the survival of patients with glioblastoma. Quantitative real-time PCR and western blot were used to evaluate mRNA and protein levels of DCBLD2. Cell viabilities were tested using Cell Counting Kit-8 and clone formation assays. Cell invasive and migratory abilities were measured by transwell assays. RESULTS: DCBLD2 expression was upregulated in glioblastoma and has a significantly positive correlation with the WHO classification. In addition, high expression of DCBLD2 was closely correlated with poor prognosis in primary and recurrent patients with glioblastoma. What is more, we found that knockdown of DCBLD2 notably reduced the cell proliferative, invasive and migratory capacities by elevating the expression of E-cadherin and inhibiting the expression of vimentin, snail, slug and twist. However, overexpression of DCBLD2 presented the opposite results. CONCLUSION: The current study reveals that high expression of DCBLD2 is closely related to poor prognosis in glioblastoma and can significantly enhance the tumor cell viability and metastasis by activating the EMT process, suggesting that DCBLD2 may be a possible biomarker for glioblastoma treatment.

MazEF Toxin-Antitoxin System-Mediated DNA Damage Stress Response in Deinococcus radiodurans.

Deinococcus radiodurans shows marked resistance to various types of DNA-damaging agents, including mitomycin C (MMC). A type II toxin-antitoxin (TA) system that responds to DNA damage stress was identified in D. radiodurans, comprising the toxin MazF-dr and the antitoxin MazE-dr. The cleavage specificity of MazF-dr, an endoribonuclease, was previously characterized. Here, we further investigated the regulatory role of the MazEF system in the response to DNA damage stress in D. radiodurans. The crystal structure of D. radiodurans MazF (MazF-dr) was determined at a resolution of 1.3 Å and is the first structure of the toxin of the TA system of D. radiodurans. MazF-dr forms a dimer mediated by the presence of interlocked loops. Transcriptional analysis revealed 650 downregulated genes in the wild-type (WT) strain, but not in the mazEF mutant strain, which are potentially regulated by MazEF-dr in response to MMC treatment. Some of these genes are involved in membrane trafficking and metal ion transportation. Subsequently, compared with the WT strain, the mazEF mutant strain exhibited much lower MMC-induced intracellular iron concentrations, reactive oxygen species (ROS), and protein carbonylation levels. These results provide evidence that MazEF-mediated cell death in D. radiodurans might be caused by an increase in ROS accumulation upon DNA damage stress.

Biochemical characterization of a unique DNA polymerase A from the extreme radioresistant organism Deinococcus radiodurans.

Deinococcus radiodurans survives extraordinary doses of ionizing radiation and desiccation that cause numerous DNA strand breaks. D. radiodurans DNA polymerase A (DrPolA) is essential for reassembling the shattered genome, while its biochemical property has not been fully demonstrated. In this study, we systematically examined the enzymatic activities of DrPolA and characterized its unique features. DrPolA contains an N-terminal nuclease domain (DrPolA-NTD) and a C-terminal Klenow fragment (KlenDr). Compared with the Klenow fragment of E. coli Pol I, KlenDr shows higher fidelity despite the lacking of 3'-5' exonuclease proofreading activity and prefers double-strand DNA rather than Primer-Template substrates. Apart from the well-annotated 5'-3' exonuclease and flap endonuclease activities, DrPolA-NTD displays approximately 140-fold higher gap endonuclease activity than its homolog in E. coli and Human FEN1. Its 5'-3' exonuclease activity on ssDNA, gap endonuclease, and Holliday junction cleavage activities are greatly enhanced by Mn2+. The DrPolA-NTD deficient strain shows increased sensitivity to UV and gamma-ray radiation. Collectively, our results reveal distinct biochemical characteristics of DrPolA during DNA degradation and re-synthesis, which provide new insight into the outstanding DNA repair capacity of D. radiodurans.

DRJAMM Is Involved in the Oxidative Resistance in Deinococcus radiodurans.

Proteins containing JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) domains that have Zn2+-dependent deubiquitinase (DUB) activity are ubiquitous across among all domains of life. Recently, a homolog in Deinococcus radiodurans, DRJAMM, was reported to possess the ability to cleave DRMoaD-MoaE. However, the detailed biochemical characteristics of DRJAMM in vitro and its biological mechanism in vivo remain unclear. Here, we show that DRJAMM has an efficient in vitro catalytic activity in the presence of Mn2+, Ca2+, Mg2+, and Ni2+ in addition to the well-reported Zn2+, and strong adaptability at a wide range of temperatures. Disruption of drJAMM led to elevated sensitivity in response to H2O2 in vivo compared to the wild-type R1. In particular, the expression level of MoaE, a product of DRJAMM cleavage, was also increased under H2O2 stress, indicating that DRJAMM is needed in the antioxidant process. Moreover, DRJAMM was also demonstrated to be necessary for dimethyl sulfoxide respiratory system in D. radiodurans. These data suggest that DRJAMM plays key roles in the process of oxidative resistance in D. radiodurans with multiple-choice of metal ions and temperatures.

MiR-1225-5p acts as tumor suppressor in glioblastoma via targeting FNDC3B.

This study attempted to research the molecular mechanism underlying the inhibitory role of miR-1225-5p in the malignant progression of glioblastoma. Bioinformatics analyses based on the gene expression omnibus (GEO) and Chinese glioma genome atlas (CGGA) databases showed that miR-1225-5p, as a favorable prognostic factor, was expressed at low levels in glioblastoma, and its expression was also related to WHO grade and age. The subsequent CCK-8 assay indicated that miR-1225-5p might prevent the malignant progression of glioblastoma, which was represented by that miR-1225-5p mimic reduced the viability of glioblastoma cells. Then, we predicted that FNDC3B might be a potential target gene of miR-1225-5p, and it was negatively correlated with the level of miR-1225-5p, which were confirmed by dual-luciferase reporter assay, qRT-PCR and western blot assays. Moreover, based on the analyses of the cancer genome atlas (TCGA), Oncomine and CGGA databases, FNDC3B was enriched in glioblastoma and high expression of FNDC3B led to poor prognosis. Finally, CCK8 and transwell experiments showed that the ability of miR-1225-5p to inhibit glioblastoma cell viability, invasion and migration was at least partially achieved by targeting FNDC3B. In general, these results revealed that the miR-1225-5p/FNDC3B axis contributes to inhibiting the malignant phenotype of glioblastoma cells, which lays a foundation for molecular diagnosis and treatment of glioblastoma.

Structural and Functional Characterization of a Unique AP Endonuclease From Deinococcus radiodurans.

Various endogenous and exogenous agents cause DNA damage, including apurinic/apyrimidinic (AP) sites. Due to their cytotoxic effects, AP sites are usually cleaved by AP endonuclease through the base excision repair (BER) pathway. Deinococcus radiodurans, an extraordinary radiation-resistant bacterium, is known as an ideal model organism for elucidating DNA repair processes. Here, we have investigated a unique AP endonuclease (DrXth) from D. radiodurans and found that it possesses AP endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-5' exonuclease but has no nucleotide incision repair (NIR) activity. We also found that Mg2+ and Mn2+ were the preferred divalent metals for endonuclease and exonuclease activities, respectively. In addition, DrXth were crystallized and the crystals diffracted to 1.5 Å. Structural and biochemical analyses demonstrated that residue Gly198 is the key residue involved in the substrate DNA binding and cleavage. Deletion of the drxth gene in D. radiodurans caused elevated sensitivity to DNA damage agents and increased spontaneous mutation frequency. Overall, our results indicate that DrXth is an important AP endonuclease involved in BER pathway and functions in conjunction with other DNA repair enzymes to maintain the genome stability.

Participation of RecJ in the base excision repair pathway of Deinococcus radiodurans.

RecJ reportedly participates in the base excision repair (BER) pathway, but structural and functional data are scarce. Herein, the Deinococcus radiodurans RecJ (drRecJ) deletion strain exhibited extreme sensitivity to hydrogen peroxide and methyl-methanesulphonate, as well as a high spontaneous mutation rate and an accumulation of unrepaired abasic sites in vivo, indicating the involvement of drRecJ in the BER pathway. The binding affinity and nuclease activity preference of drRecJ toward DNA substrates containing a 5'-P-dSpacer group, a 5'-deoxyribose-phosphate (dRP) mimic, were established. A 1.9 Å structure of drRecJ in complex with 5'-P-dSpacer-modified single-stranded DNA (ssDNA) revealed a 5'-monophosphate binding pocket and occupancy of 5'-dRP in the drRecJ nuclease core. The mechanism for RecJ 5'-dRP catalysis was explored using structural and biochemical data, and the results implied that drRecJ is not a canonical 5'-dRP lyase. Furthermore, in vitro reconstitution assays indicated that drRecJ tends to participate in the long-patch BER pathway rather than the short-patch BER pathway.

Succinylation at a key residue of FEN1 is involved in the DNA damage response to maintain genome stability.

Human flap endonuclease 1 (FEN1) is a structure-specific, multifunctional endonuclease essential for DNA replication and repair. Our previous study showed that in response to DNA damage, FEN1 interacts with the PCNA-like Rad9-Rad1-Hus1 complex instead of PCNA to engage in DNA repair activities, such as stalled DNA replication fork repair, and undergoes SUMOylation by SUMO-1. Here, we report that succinylation of FEN1 was stimulated in response to DNA replication fork-stalling agents, such as ultraviolet (UV) irradiation, hydroxyurea, camptothecin, and mitomycin C. K200 is a key succinylation site of FEN1 that is essential for gap endonuclease activity and could be suppressed by methylation and stimulated by phosphorylation to promote SUMO-1 modification. Succinylation at K200 of FEN1 promoted the interaction of FEN1 with the Rad9-Rad1-Hus1 complex to rescue stalled replication forks. Impairment of FEN1 succinylation led to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of FEN1 succinylation in regulating its roles in DNA replication and repair, thus maintaining genome stability.

Late embryogenesis abundant group3 protein (DrLEA3) is involved in antioxidation in the extremophilic bacterium Deinococcus radiodurans.

Deinococcus radiodurans is able to survive under extreme conditions, including high doses of ionizing radiation, desiccation and oxidative stress. In addition to enhanced DNA repair capabilities, an effective antioxidation system plays an important role in its robustness. Previous studies have linked the radiation resistance of D. radiodurans to its prolonged desiccation tolerance phenotype, which both cause DNA damage. In the current study, we investigated the roles of dr_1172 in D. radiodurans, the gene encoding a typical group 3 LEA protein (DrLEA3) conserved within Deinococcus species. In addition to the increased transcriptional level under oxidative stress, the inactivation of dr_1172-sensitized cells to H2O2 treatments and the reduced cellular antioxidation activities suggested that dr_1172 is involved in the cellular defense against oxidative stress. Moreover, DrLEA3 was enriched at the cell membrane and bound to various types of metal ions. Cells devoid of DrLEA3 showed a decreased intracellular Mn/Fe concentration ratio, indicating that DrLEA3 also plays a role in maintaining metal ion homeostasis in vivo.

N 4-Cytosine DNA Methylation Is Involved in the Maintenance of Genomic Stability in Deinococcus radiodurans.

DNA methylation serves as a vital component of restriction-modification (R-M) systems in bacteria, where it plays a crucial role in defense against foreign DNA. Recent studies revealed that DNA methylation has a global impact on gene expression. Deinococcus radiodurans, an ideal model organism for studying DNA repair and genomic stability, possesses unparalleled resistance to DNA-damaging agents such as irradiation and strong oxidation. However, details on the methylome of this bacterium remain unclear. Here, we demonstrate that N 4-cytosine is the major methylated form (4mC) in D. radiodurans. A novel methylated motif, "C4mCGCGG" was identified that was fully attributed to M.DraR1 methyltransferase. M.DraR1 can specifically bind and methylate the second cytosine at N 4 atom of "CCGCGG" motif, preventing its digestion by a cognate restriction endonuclease. Cells deficient in 4mC modification displayed higher spontaneous rifampin mutation frequency and enhanced DNA recombination and transformation efficiency. And genes involved in the maintenance of genomic stability were differentially expressed in conjunction with the loss of M.DraR1. This study provides evidence that N 4-cytosine DNA methylation contributes to genomic stability of D. radiodurans and lays the foundation for further research on the mechanisms of epigenetic regulation by R-M systems in bacteria.

Succinylome Analysis Reveals the Involvement of Lysine Succinylation in the Extreme Resistance of Deinococcus radiodurans.

Increasing evidence shows that the succinylation of lysine residues mainly regulates enzymes involved in the carbon metabolism pathway, in both prokaryotic and eukaryotic cells. Deinococcus radiodurans is one of the most radioresistant organisms on earth and is famous for its robust resistance. A major goal in the current study of protein succinylation is to explore its function in D. radiodurans. High-resolution LC-MS/MS is used for qualitative proteomics to perform a global succinylation analysis of D. radiodurans and 492 succinylation sites in 270 proteins are identified. These proteins are involved in a variety of biological processes and pathways. It is found that the enzymes involved in nucleic acid binding/processing are enriched in D. radiodurans compared with their previously reported levels in other bacteria. The mutagenesis studies confirm that succinylation regulates the enzymatic activities of species-specific proteins PprI and DdrB, which belong to the radiation-desiccation response regulon. Together, these results provide insight into the role of lysine succinylation in the extreme resistance of D. radiodurans.

Structure and DNA damage-dependent derepression mechanism for the XRE family member DG-DdrO.

DdrO is an XRE family transcription repressor that, in coordination with the metalloprotease PprI, is critical in the DNA damage response of Deinococcus species. Here, we report the crystal structure of Deinococcus geothermalis DdrO. Biochemical and structural studies revealed the conserved recognizing α-helix and extended dimeric interaction of the DdrO protein, which are essential for promoter DNA binding. Two conserved oppositely charged residues in the HTH motif of XRE family proteins form salt bridge interactions that are essential for promoter DNA binding. Notably, the C-terminal domain is stabilized by hydrophobic interactions of leucine/isoleucine-rich helices, which is critical for DdrO dimerization. Our findings suggest that DdrO is a novel XRE family transcriptional regulator that forms a distinctive dimer. The structure also provides insight into the mechanism of DdrO-PprI-mediated DNA damage response in Deinococcus.