Bing Tian

PprI : a general switch responsible for extreme radioresistance of Deinococcus radiodurans

Deinococcus radiodurans exhibits an extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents, particularly, ionizing radiation. Available evidence indicates that efficient repair of DNA damage and protection of the chromosomal structure are mainly responsible for the radioresistance. Little is known about the biochemical basis for this phenomenon. We have identified a unique gene, pprI, as a general switch for downstream DNA repair and protection pathways, from a natural mutant, in which pprI is disrupted by a transposon. Complete functional disruption of the gene in wild-type leads to dramatic sensitivity to ionizing radiation. Radioresistance of the disruptant could be fully restored by complementation with pprI. In response to radiation stress, PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection pathways in response to radiation stress.

A Novel OxyR Sensor and Regulator of Hydrogen Peroxide Stress with One Cysteine Residue in Deinococcus radiodurans

In bacteria, OxyR is a peroxide sensor and transcription regulator, which can sense the presence of reactive oxygen species and induce antioxidant system. When the cells are exposed to H2O2, OxyR protein is activated via the formation of a disulfide bond between the two conserved cysteine residues (C199 and C208). In Deinococcus radiodurans, a previously unreported special characteristic of DrOxyR (DR0615) is found with only one conserved cysteine. dr0615 gene mutant is hypersensitive to H2O2, but only a little to ionizing radiation. Site-directed mutagenesis and subsequent in vivo functional analyses revealed that the conserved cysteine (C210) is necessary for sensing H2O2, but its mutation did not alter the binding characteristics of OxyR on DNA. Under oxidant stress, DrOxyR is oxidized to sulfenic acid form, which can be reduced by reducing reagents. In addition, quantitative real-time PCR and global transcription profile results showed that OxyR is not only a transcriptional activator (e.g., katE, drb0125), but also a transcriptional repressor (e.g., dps, mntH). Because OxyR regulates Mn and Fe ion transporter genes, Mn/Fe ion ratio is changed in dr0615 mutant, suggesting that the genes involved in Mn/Fe ion homeostasis, and the genes involved in antioxidant mechanism are highly cooperative under extremely oxidant stress. In conclusion, these findings expand the OxyR family, which could be divided into two classes: typical 2-Cys OxyR and 1-Cys OxyR.

DrRRA: a novel response regulator essential for the extreme radioresistance of Deinococcus radiodurans

Two‐component systems are predominant signal transduction pathways in prokaryotes, and also exist in many archaea as well as some eukaryotes. A typical TCS consists of a histidine kinase and a cognate response regulator. In this study, a novel gene encoding a response regulator (we designate it drRRA) is identified to be essential for the extreme radioresistance of Deinococcus radiodurans. DrRRA null mutant (we designate it MR) is sensitive to gamma‐radiation compared with the wild‐type strain. Transcriptional assays show that numerous genes are changed in their transcriptional levels in MR at exponential growth phase under normal or gamma‐radiation condition. Most of them are related to stress response and DNA repair. Antioxidant activity assays exhibit that both superoxide dismutases and catalases are decreased in the mutant, whereas Western blotting assays show that RecA and PprA are also reduced in MR, verifying the microarray and quantitative real‐time PCR data. Furthermore, pulsed‐field gel electrophoresis assay demonstrates that deletion of drRRA results in the delay of genome restitution. These data support the hypothesis that DrRRA contributes to the extreme radioresistance of D. radiodurans through its regulatory role in multiple pathways such as antioxidation and DNA repair pathways.

Effects of carotenoids from Deinococcus radiodurans on protein oxidation

Aims:  To evaluate the antioxidant effect of carotenoids from Deinococcus radiodurans on protein.

DdrB stimulates single-stranded DNA annealing and facilitates RecA-independent DNA repair in Deinococcus radiodurans

The bacterium Deinococcus radiodurans can survive extremely high exposure to ionizing radiation. The repair mechanisms involved in this extraordinary ability are still being investigated. ddrB is one gene that is highly up-regulated after irradiation, and it has been proposed to be involved in RecA-independent repair in D. radiodurans. Here we cloned, expressed and characterized ddrB in order to define its roles in the radioresistance of D. radiodurans. DdrB preferentially binds to single-stranded DNA. Moreover, it interacts directly with single-stranded binding protein of D. radiodurans DrSSB, and stimulates single-stranded DNA annealing even in the presence of DrSSB. The post-irradiation DNA repair kinetics of a ddrB/recA double mutant were compared to ddrB and recA single mutants by pulsed-field gel electrophoresis (PFGE). DNA fragment rejoining in the ddrB/recA double mutant is severely compromised, suggesting that DdrB-mediated single-stranded annealing plays a critical role in the RecA-independent DNA repair of D. radiodurans.

RecO Is Essential for DNA Damage Repair in Deinococcus radiodurans

Here we present direct evidence for the vital role of RecO in Deinococcus radioduranss radioresistance. A recO null mutant was constructed using a deletion replacement method. The mutant exhibited a growth defect and extreme sensitivity to irradiation with gamma rays and UV light. These results suggest that DNA repair in this organism occurs mainly via the RecF pathway.

Identification and evaluation of the role of the manganese efflux protein in Deinococcus radiodurans

BackgroundDeinococcus radiodurans accumulates high levels of manganese ions, and this is believed to be correlated with the radiation resistance ability of this microorganism. However, the maintenance of manganese ion homeostasis in D. radiodurans remains to be investigated.ResultsIn this study, we identified the manganese efflux protein (MntE) in D. radiodurans. The null mutant of mntE was more sensitive than the wild-type strain to manganese ions, and the growth of the mntE mutant was delayed in manganese-supplemented media. Furthermore, there was a substantial increase in the in vivo concentration of manganese ions. Consistent with these characteristics, the mntE mutant was more resistant to H2O2, ultraviolet rays, and γ-radiation. The intracellular protein oxidation (carbonylation) level of the mutant strain was remarkably lower than that of the wild-type strain.ConclusionsOur results indicated that dr1236 is indeed a mntE homologue and is indispensable for maintaining manganese homeostasis in D. radiodurans. The data also provide additional evidence for the involvement of intracellular manganese ions in the radiation resistance of D. radiodurans.

DR2539 is a novel DtxR-like regulator of Mn/Fe ion homeostasis and antioxidant enzyme in Deinococcus radiodurans

Transcriptional regulators of the diphtheria toxin repressor (DtxR) family control the expression of genes involved in the uptake of iron and manganese, which is not only necessitous nutrients but also was suggested to be essential for intracellular redox cycling of microorganisms. We identified a unique DtxR homologue (DR2539) with special characteristics from Deinococcus radiodurans, which is known for its extreme resistance to radiation and oxidants. The dr2539 mutant showed higher resistance to hydrogen peroxide than the wild-type strain R1. Intracellular catalase activity assay and semiquantitative PCR analysis demonstrated that this DtxR is a negative regulator of catalase (katE). Furthermore, quantitative real-time PCR, global transcription profile and inductively coupled plasma-mass spectrometry analysis showed that the DtxR is involved in the regulation of antioxidant system by maintaining the intracellular Mn/Fe ion homeostasis of D. radiodurans. However, unlike the other DtxR homologues, the DtxR of D. radiodurans acts as a negative regulator of a Mn transporter gene (dr2283) and as a positive regulator of Fe-dependent transporter genes (dr1219, drb0125) in D. radiodurans.

The Essential Role of H19 Contributing to Cisplatin Resistance by Regulating Glutathione Metabolism in High-Grade Serous Ovarian Cancer.

Primary and acquired drug resistance is one of the main obstacles encountered in high-grade serous ovarian cancer (HGSC) chemotherapy. Cisplatin induces DNA damage through cross-linking and long integrated non-coding RNAs (lincRNAs) play an important role in chemical induced DNA-damage response, which suggests that lincRNAs may be also associated with cisplatin resistance. However, the mechanism of long integrated non-coding RNAs (lincRNAs) acting on cisplatin resistance is not well understood. Here, we showed that expression of lin-RECK-3, H19, LUCAT1, LINC00961, and linc-CARS2-2 was enhanced in cisplatin-resistant A2780-DR cells, while transcriptome sequencing showed decreased Linc-TNFRSF19-1 and LINC00515 expression. Additionally, we verified that different H19 expression levels in HGSC tissues showed strong correlation with cancer recurrence. H19 knockdown in A2780-DR cells resulted in recovery of cisplatin sensitivity in vitro and in vivo. Quantitative proteomics analysis indicated that six NRF2-targeted proteins, including NQO1, GSR, G6PD, GCLC, GCLM and GSTP1 involved in the glutathione metabolism pathway, were reduced in H19-knockdown cells. Furthermore, H19-knockdown cells were markedly more sensitive to hydrogen-peroxide treatment and exhibited lower glutathione levels. Our results reveal a previously unknown link between H19 and glutathione metabolism in the regulation of cancer-drug resistance.

Structural basis for DNA 5´-end resection by RecJ

The resection of DNA strand with a 5´ end at double-strand breaks is an essential step in recombinational DNA repair. RecJ, a member of DHH family proteins, is the only 5´ nuclease involved in the RecF recombination pathway. Here, we report the crystal structures of Deinococcus radiodurans RecJ in complex with deoxythymidine monophosphate (dTMP), ssDNA, the C-terminal region of single-stranded DNA-binding protein (SSB-Ct) and a mechanistic insight into the RecF pathway. A terminal 5´-phosphate-binding pocket above the active site determines the 5´-3´ polarity of the deoxy-exonuclease of RecJ; a helical gateway at the entrance to the active site admits ssDNA only; and the continuous stacking interactions between protein and nine nucleotides ensure the processive end resection. The active site of RecJ in the N-terminal domain contains two divalent cations that coordinate the nucleophilic water. The ssDNA makes a 180° turn at the scissile phosphate. The C-terminal domain of RecJ binds the SSB-Ct, which explains how RecJ and SSB work together to efficiently process broken DNA ends for homologous recombination. DOI: http://dx.doi.org/10.7554/eLife.14294.001