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.

POLM variant G312R promotes ovarian tumorigenesis through genomic instability and COL11A1-NF-κB axis.

In ovarian cancer (OC), identifying key molecular players in disease escalation and chemoresistance remains critical. Our investigation elucidates the role of the DNA polymerase mu (POLM), especially G312R mutation, in propelling oncogenesis through dual pathways. POLMG312R markedly augments the ribonucleotide insertion capability of POLM, precipitating genomic instability. In addition, our research reveals that POLMG312R perturbs collagen alpha-1 (XI) chain (COL11A1) expression-a gene that plays a key role in oncogenesis-and modulates the NF-κB signaling pathway, alters the secretion of downstream inflammatory cytokines, and promotes tumor-macrophage interactions. We illustrate a bidirectional regulatory interaction between POLM, particularly its G312R variant, and COL11A1. This interaction regulates NF-κB signaling, culminating in heightened malignancy and resistance to chemotherapy in OC cells. These insights position the POLM as a potential molecular target for OC therapy, shedding light on the intricate pathways underpinning POLM variant disease progression.NEW & NOTEWORTHY Our research reveals that POLM plays an important role in ovarian cancer development, especially the mutation G312R. We uncover the POLMG312R mutation as a driver of genomic instability in ovarian cancer via aberrant ribonucleotide incorporation. We reveal that POLMG312R upregulates COL11A1 and activates NF-κB signaling, contributing to tumor progression and chemoresistance. This study identifies the POLM-COL11A1-NF-κB axis as a novel oncogenic pathway.

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.

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.

N4 -Allyldeoxycytidine: A New DNA Tag with Chemical Sequencing Power for Pinpointing Labelling Sites, Mapping Epigenetic Markers, and In Situ Imaging.

DNA tagging with base analogues has found numerous applications. To precisely record the DNA labelling information, it would be highly beneficial to develop chemical sequencing tags that can be encoded into DNA as regular bases and decoded as mutant bases following a mild, efficient and bioorthogonal chemical treatment. Here we reported such a DNA tag, N4 -allyldeoxycytidine (a4 dC), for labeling and identifying DNA by in vitro assays. The iodination of a4 dC led to fast and complete formation of 3, N4 -cyclized deoxycytidine, which induced base misincorporation during DNA replication and thus could be located at single base resolution. We explored the applications of a4 dC in pinpointing DNA labelling sites at single base resolution, mapping epigenetic marker N4 -methyldeoxycytidine, and imaging nucleic acids in situ. In addition, mammalian cellular DNA could be metabolically labelled with a4 dC. Our study sheds light on the design of next generation DNA tags with chemical sequencing power.

Distinct lipid membrane interaction and uptake of differentially charged nanoplastics in bacteria.

BACKGROUND: Nanoplastics have been recently found widely distributed in our natural environment where ubiquitously bacteria are major participants in various material cycles. Understanding how nanoplastics interact with bacterial cell membrane is critical to grasp their uptake processes as well as to analyze their associated risks in ecosystems and human microflora. However, little is known about the detailed interaction of differentially charged nanoplastics with bacteria. The present work experimentally and theoretically demonstrated that nanoplastics enter into bacteria depending on the surface charges and cell envelope structural features, and proved the shielding role of membrane lipids against nanoplastics. RESULTS: Positively charged polystyrene nanoplastics (PS-NH2, 80 nm) can efficiently translocate across cell membranes, while negatively charged PS (PS-COOH) and neutral PS show almost no or much less efficacy in translocation. Molecular dynamics simulations revealed that the PS-NH2 displayed more favourable electrostatic interactions with bacterial membranes and was subjected to internalisation through membrane penetration. The positively charged nanoplastics destroy cell envelope of Gram-positive B. subtilis by forming membrane pore, while enter into the Gram-negative E. coli with a relatively intact envelope. The accumulated positively charged nanoplastics conveyed more cell stress by inducing a higher level of reactive oxygen species (ROS). However, the subsequently released membrane lipid-coated nanoplastics were nearly nontoxic to cells, and like wise, stealthy bacteria wrapped up with artifical lipid layers became less sensitive to the positively charged nanoplastics, thereby illustrating that the membrane lipid can shield the strong interaction between the positively charged nanoplastics and cells. CONCLUSIONS: Our findings elucidated the molecular mechanism of nanoplastics' interaction and accumulation within bacteria, and implied the shielding and internalization effect of membrane lipid on toxic nanoplastics could promote bacteria for potential plastic bioremediation.

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.

Dynamic Polyphosphate Metabolism Coordinating with Manganese Ions Defends against Oxidative Stress in the Extreme Bacterium Deinococcus radiodurans.

Deinococcus radiodurans is an extreme bacterium with unparalleled resistance to oxidative stresses. Accumulation of intracellular Mn2+ complexing with small metabolites is the key contributor to the tolerance of D. radiodurans against oxidative stress. However, the intracellular reservoir of Mn ions and homeostatic regulation of the Mn complex in D. radiodurans remain unclear. We identified an evolutionarily ancient and negatively charged phosphate polymer (polyphosphate [PolyP]) in D. radiodurans We investigated PolyP metabolism in the response of D. radiodurans to oxidative stress. The genes dr1939, encoding polyphosphatase kinase (PPKDr; the subscript "Dr" refers to D. radiodurans), and dra0185, encoding exopolyphosphatase (PPXDr), were identified. PPXDr is a novel exopolyphosphatase with a cofactor preference to Mn2+, which enhances the dimerization and activity of PPXDr to allow the effective cleavage of PolyP-Mn. PPKDr and PPXDr exhibited different dynamic expression profiles under oxidative stress. First, ppkDr was upregulated leading to the accumulation of PolyP, which chelated large amounts of intracellular Mn ions. Subsequently, the expression level of ppkDr decreased while ppxDr was substantially upregulated and effectively hydrolyzed inactive PolyP-Mn to release phosphate (Pi) and Mn2+, which could form into Mn-Pi complexes to scavenge O2- and protect proteins from oxidative damage. Hence, dynamic cellular PolyP metabolites complexed with free Mn ions highlight a defense strategy of D. radiodurans in response to oxidative stress.IMPORTANCE The Mn-phosphate complex (Mn-Pi) plays a key role in the cellular resistance of radioresistant bacteria. The evolutionarily ancient polyphosphate polymers (polyphosphate [PolyP]) could effectively chelate Mn2+ and donate phosphates. However, the intracellular reservoir of Mn ions and homeostatic regulation of the Mn-Pi complex remain unclear. Here, we investigated the relationship of PolyP metabolites and Mn2+ homeostasis and how they function to defend against oxidative stress in the radioresistant bacterium Deinococcus radiodurans We found that PPXDr (the subscript "Dr" refers to D. radiodurans) is a novel exopolyphosphatase with a cofactor preference for Mn2+, mediating PolyP-Mn degradation into Pi and Mn ions. The formed Mn-Pi complexes effectively protect proteins. The dynamic PolyP metabolism coordinating with Mn ions is a defense strategy of D. radiodurans in response to oxidative stress. The findings not only provide new insights into the resistance mechanism of the extreme bacterium D. radiodurans but also broaden our understanding of the functions of PolyP metabolism in organisms.

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.

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.

Structural basis of 5' flap recognition and protein-protein interactions of human flap endonuclease 1.

Human flap endonuclease 1 (hFEN1) is a structure-specific nuclease essential for DNA replication and repair processes. hFEN1 has 5' flap removal activity, as well as gap endonuclease activity that is critical for restarting stalled replication forks. Here, we report the crystal structures of wild-type and mutant hFEN1 proteins in complex with DNA substrates, followed by mutagenesis studies that provide mechanistic insight into the protein-protein interactions of hFEN1. We found that in an α-helix forming the helical gateway of hFEN1 recognizes the 5' flap prior to its threading into the active site for cleavage. We also found that the ß-pin region is rigidified into a short helix in R192F hFEN1-DNA structures, suppressing its gap endonuclease activity and cycle-dependent kinase interactions. Our findings suggest that a single mutation at the primary methylation site can alter the function of hFEN1 and provide insight into the role of the ß-pin region in hFEN1 protein interactions that are essential for DNA replication and repair.

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.

Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex.

During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5' ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.

Distinct roles of Deinococcus radiodurans RecFOR and RecA in DNA transformation.

RecFOR and RecA are key recombination factors in Deinococcus radiodurans, a bacterium that possesses robust DNA repair capability and is also naturally transformable. While RecFOR functioning as a RecA loader during DNA repair has been established, their relative roles in transformation need further exploration. Here, we constructed recFOR and recA deletion mutants of D. radiodurans, and investigated the effect of these mutations on DNA transformation. recA deletion causes defects in both plasmid and chromosomal transformation. However, it was found that recFOR is not involved in chromosomal transformation, and that only recO and recR mutations compromise plasmid transformation. How recO, recR and recA mutations influence plasmid transformation was further examined by complementation plasmids. Interestingly, the transformation process remains defective in the recA mutant, but gets restored in the recO and recR mutants. This indicates that unlike RecA, RecOR may not be essential for DNA uptake. Therefore, we provide evidence that RecFOR is dispensable for RecA to protect incoming exogenous DNA and to catalyze recombination during transformation. Instead, RecO and RecR are likely to promote later steps in plasmid transformation.

Deinococcus radiodurans DR1088 is a novel RecF-interacting protein that stimulates single-stranded DNA annealing.

RecF, together with the recombination mediators RecO and RecR, is required in the RecFOR homologous recombination repair pathway in bacteria. In this study, a recF-dr1088 operon, which is highly conserved in the Deinococcus-Thermus phylum, was identified in Deinococcus radiodurans. Interaction between DRRecF and DR1088 was confirmed by yeast two-hybrid and pull-down assays. DR1088 exhibited some RecO-like biochemical properties including single/double-stranded DNA binding activity, ssDNA binding protein (SSB) replacement ability and ssDNA (with or without SSB) annealing activity. However, unlike other recombination proteins, dr1088 is essential for cell viability. These results indicate that DR1088 might play a role in DNA replication and DNA repair processes.

Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.

Flap endonuclease 1 (FEN1) phosphorylation is proposed to regulate the action of FEN1 in DNA repair as well as Okazaki fragment maturation. However, the biologic significance of FEN1 phosphorylation in response to DNA damage remains unknown. Here, we report an in vivo role for FEN1 phosphorylation, using a mouse line carrying S187A FEN1, which abolishes FEN1 phosphorylation. Although S187A mouse embryonic fibroblast cells showed normal proliferation under low oxygen levels (2%), the mutant cells accumulated oxidative DNA damage, activated DNA damage checkpoints, and showed G1-phase arrest at atmospheric oxygen levels (21%). This suggests an essential role for FEN1 phosphorylation in repairing oxygen-induced DNA damage and maintaining proper cell cycle progression. Consistently, the mutant cardiomyocytes showed G1-phase arrest due to activation of the p53-mediated DNA damage response at the neonatal stage, which reduces the proliferation potential of the cardiomyocytes and impairs heart development. Nearly 50% of newborns with the S187A mutant died in the first week due to failure to undergo the peroxisome proliferator-activated receptor signaling-dependent switch from glycolysis to fatty acid oxidation. The adult mutant mice developed dilated hearts and showed significantly shorter life spans. Altogether, our results reveal an important role of FEN1 phosphorylation to counteract oxygen-induced stress in the heart during the fetal-to-neonatal transition.-Zhou, L., Dai, H., Wu, J., Zhou, M., Yuan, H., Du, J., Yang, L., Wu, X., Xu, H., Hua, Y., Xu, J., Zheng, L., Shen, B. Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.

Watching DNA polymerase η make a phosphodiester bond.

DNA synthesis has been extensively studied, but the chemical reaction itself has not been visualized. Here we follow the course of phosphodiester bond formation using time-resolved X-ray crystallography. Native human DNA polymerase η, DNA and dATP were co-crystallized at pH 6.0 without Mg(2+). The polymerization reaction was initiated by exposing crystals to 1 mM Mg(2+) at pH 7.0, and stopped by freezing at desired time points for structural analysis. The substrates and two Mg(2+) ions are aligned within 40 s, but the bond formation is not evident until 80 s. From 80 to 300 s structures show a mixture of decreasing substrate and increasing product of the nucleotidyl-transfer reaction. Transient electron densities indicate that deprotonation and an accompanying C2'-endo to C3'-endo conversion of the nucleophile 3'-OH are rate limiting. A third Mg(2+) ion, which arrives with the new bond and stabilizes the intermediate state, may be an unappreciated feature of the two-metal-ion mechanism.

DqsIR quorum sensing-mediated gene regulation of the extremophilic bacterium Deinococcus radiodurans in response to oxidative stress.

Here, we show that AHLs can be employed by Deinococcus radiodurans, which belongs to the unique phylum Deinococcus-Thermus and is known for its cellular resistance to environmental stresses. An AHL-mediated quorum-sensing system (DqsI/DqsR) was identified in D. radiodurans. We found that under non-stress conditions, the AHL level was "shielded" by quorum quenching enzymes, whereas AHLs accumulated when D. radiodurans was exposed to oxidative stress. Upon exposure to H2 O2 , AHL synthetic enzymes (DqsI) were immediately induced, while the expression of quorum-quenching enzymes began to increase approximately 30 min after exposure to H2 O2 , as shown by time-course analyses of gene expression. Both dqsI mutant (DMDqsI) and dqsR mutant (MDqsR) were more sensitive to oxidative stress compared with the wild-type strain. Exogenous AHLs (5 µM) could completely restore the survival fraction of DMDqsI under oxidative stress. RNA-seq analysis showed that a number of genes involved in stress-response, cellular cleansing, and DNA repair had altered transcriptional levels in MDqsR. The DqsR, acting as a regulator of quorum sensing, controls gene expression along with AHLs. Hence, the DqsIR-mediated quorum sensing that mediates gene regulation is an adaptive strategy for D. radiodurans in response to oxidative stresses and is conserved in the extremophilic Deinococcus bacteria.

Structural insights into catalysis and dimerization enhanced exonuclease activity of RNase J.

RNase J is a conserved ribonuclease that belongs to the ß-CASP family of nucleases. It possesses both endo- and exo-ribonuclease activities, which play a key role in pre-rRNA maturation and mRNA decay. Here we report high-resolution crystal structures of Deinococcus radiodurans RNase J complexed with RNA or uridine 5'-monophosphate in the presence of manganese ions. Biochemical and structural studies revealed that RNase J uses zinc ions for two-metal-ion catalysis. One residue conserved among RNase J orthologues (motif B) forms specific electrostatic interactions with the scissile phosphate of the RNA that is critical for the catalysis and product stabilization. The additional manganese ion, which is coordinated by conserved residues at the dimer interface, is critical for RNase J dimerization and exonuclease activity. The structures may also shed light on the mechanism of RNase J exo- and endonucleolytic activity switch.

Characterization and role of a 2',3'-cyclic phosphodiesterase from Deinococcus radiodurans.

OBJECTIVES: A 2',3'-cyclic phosphodiesterase gene (drCPDase) has been characterized from Deinococcus radiodurans and is involved in the robust resistance of this organism. RESULTS: Cells lacking 2',3'-cyclic phosphodiesterase gene (drCPDase) showed modest growth defects and displayed increased sensitivities to high doses of various DNA-damaging agents including ionizing radiation, mitomycin C, UV and H2O2. The transcriptional level of drCPDase increased after H2O2 treatment. Additional nucleotide monophosphate partially recovered the phenotype of drCPDase knockout cells. Complementation of E. coli with drCPDase resulted in enhanced H2O2 resistance. CONCLUSIONS: The 2',3'-cyclic phosphodiesterase (drCPDase) contributes to the extreme resistance of D. radiodurans and is presumably involved in damaged nucleotide detoxification.