Lianrong Wang

Wuhan University, Wu-han-shih, Hubei, China

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Publications (15)68.44 Total impact

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    ABSTRACT: The modification of DNA by phosphorothioate (PT) occurs when the non-bridging oxygen in the sugar-phosphate backbone of DNA is replaced with sulfur. This DNA backbone modification was recently discovered and is governed by the dndABCDE genes in a diverse group of bacteria and archaea. However, the biological function of DNA PT modifications is poorly understood. In this study, we employed the RNA-seq analysis to characterize the global transcriptional changes in response to PT modifications. Our results show that DNA without PT protection is susceptible to DNA damage caused by the dndFGHI gene products. The DNA double-stranded breaks then trigger the SOS response, cell filamentation and prophage induction. Heterologous expression of dndBCDE conferring DNA PT modifications at GPSA and GPST prevented the damage in Salmonella enterica. Our data provide insights into the physiological role of the DNA PT system.
    Scientific reports. 01/2014; 4:6642.
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    ABSTRACT: DNA phosphorothioate (PT) modification is a recently identified epigenetic modification that occurs in the sugar-phosphate backbone of prokaryotic DNA. Previous studies have demonstrated that DNA PT modification is governed by the five DndABCDE proteins in a sequence-selective and RP stereo-specific manner. Bacteria may have acquired this physiological modification along with dndFGH as a restriction-modification system. However, little is known about the biological function of Dnd proteins, especially the smallest protein, DndE, in the PT modification pathway. DndE was reported to be a DNA-binding protein with a preference for nicked dsDNA in vitro; the binding of DndE to DNA occurs via six positively charged lysine residues on its surface. The substitution of these key lysine residues significantly decreased the DNA binding affinities of DndE proteins to undetectable levels. In this study, we conducted site-directed mutagenesis of dndE on a plasmid and measured DNA PT modifications under physiological conditions by mass spectrometry. We observed distinctive differences from the in vitro binding assays. Several mutants with lysine residues mutated to alanine decreased the total frequency of PT modifications, but none of the mutants completely eliminated PT modification. Our results suggest that the nicked dsDNA-binding capacity of DndE may not be crucial for PT modification and/or that DndE may have other biological functions in addition to binding to dsDNA.
    PLoS ONE 01/2014; 9(9):e107981. · 3.53 Impact Factor
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    ABSTRACT: Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of GpsAAC/GpsTTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at CpsCA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms.
    Nature Communications 01/2014; 5:3951. · 10.74 Impact Factor
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    Lianrong Wang, Shi Chen, Zixin Deng
    08/2011; , ISBN: 978-953-307-593-8
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    ABSTRACT: Phosphorothioate (PT) modification of DNA, with sulfur replacing a nonbridging phosphate oxygen, was recently discovered as a product of the dnd genes found in bacteria and archaea. Given our limited understanding of the biological function of PT modifications, including sequence context, genomic frequencies, and relationships to the diversity of dnd gene clusters, we undertook a quantitative study of PT modifications in prokaryotic genomes using a liquid chromatography-coupled tandem quadrupole mass spectrometry approach. The results revealed a diversity of unique PT sequence contexts and three discrete genomic frequencies in a wide range of bacteria. Metagenomic analyses of PT modifications revealed unique ecological distributions, and a phylogenetic comparison of dnd genes and PT sequence contexts strongly supports the horizontal transfer of dnd genes. These results are consistent with the involvement of PT modifications in a type of restriction-modification system with wide distribution in prokaryotes.
    Proceedings of the National Academy of Sciences 02/2011; 108(7):2963-8. · 9.81 Impact Factor
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    ABSTRACT: The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1'-oxidation and the nucleoside 5'-aldehyde of 5'-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5'-aldehyde lesions. Further, the well-defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin gamma(1)(I), was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5'-aldehyde per 10(6) nt per microM in accord with its established minor 1'- and major 5'-oxidation chemistry. Calicheamicin unexpectedly caused 1'-oxidation at a low level of 10 2-deoxyribonolactone per 10(6) nt per microM in addition to the expected predominance of 5'-oxidation at 560 nucleoside 5'-aldehyde per 10(6) nt per microM. The two hydroxyl radical-mediated DNA oxidants, gamma-radiation and Fe(2+)-EDTA, produced nucleoside 5'-aldehyde at a frequency of 57 per 10(6) nt per Gy (G-value 74 nmol/J) and 3.5 per 10(6) nt per microM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, gamma-radiation and Fe(2+)-EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 10(6) nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 10(6) nt per microM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5'-aldehyde of 9.7 and 73 lesions per 10(6) nt, respectively. Gamma-irradiation of the cells caused increases of 0.045 and 0.22 lesions per 10(6) nt per Gy, respectively, which represents a approximately 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by gamma-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between gamma-radiation and Fe(2+)-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.
    Journal of the American Chemical Society 04/2010; 132(17):6145-53. · 10.68 Impact Factor
  • Shi Chen, Lianrong Wang, Zixin Deng
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    ABSTRACT: Here we tell a 20-year long story. It began with an easily overlooked DNA degradation (Dnd) phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by five dnd genes. This unusual DNA modification, called phosphorothioation, is the first physiological modification identified on the DNA backbone, in which the nonbridging oxygen is replaced by sulfur in a sequence selective and stereo-specific manner. Homologous dnd gene clusters have been identified in diverse and distantly related bacteria and thus have drawn immediate attention of the entire microbial scientific community. Here, we summarize the progress in chemical, genetic, enzymatic, bioinformatical and analytical aspects of this novel postreplicative DNA modification. We also discuss perspectives on the physiological functions of the DNA phosphorothioate modification in bacteria and their implications.
    Protein & Cell 01/2010; 1(1):14-21. · 3.22 Impact Factor
  • Lianrong Wang, Dr Koli Taghizadeh
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    ABSTRACT: Supervisor's supporting comments I fully support Lianrong Wang's nomination as a Bioanalysis Young Investigator. Over the course of a few years, she has learned a tremendous amount about bioanalytical chemistry and technology, which has greatly broadened her perspective on microbiology and biomedical science in general. She quickly mastered a wide range of both chromatographic and mass spectrometric methods, which she systematically applied to solve a long-standing problem in microbiology: the question of the chemical identity of sulfur incorporated into DNA in bacteria possessing the dnd gene cluster. Her efforts led to the very important discovery of phosphorothioate DNA modifications as natural products, demonstrating the power of applying bioanalysis to biological problems. Lianrong Wang now possesses a valuable interdisciplinary skill set that will allow her to tackle important problems in drug development and biomedical science.
    Bioanalysis 09/2009; 1(6):1041-1042. · 3.25 Impact Factor
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    ABSTRACT: A novel DNA phosphorothioate modification (DNA sulfur modification), in which one of the non-bridging oxygen atoms in the phosphodiester bond linking DNA nucleotides is exchanged by sulphur, was found to be genetically determined by dnd or dnd-counterpart loci in a wide spectrum of bacteria from diverse habitats. A detailed mutational analysis of the individual genes within the dnd locus in Streptomyces lividans responsible for DNA phosphorothioation was performed and is described here. It should be of great help for the mechanistic study of this intriguing system. A 6,665-bp DNA region carrying just five ORFs (dndA-E) was defined as the sole determinant for modification of the DNA backbone in S. lividans to form phosphorothioate. This provides a diagnostically reliable and easily assayable Dnd (DNA degradation) phenotype. While dndA is clearly transcribed independently, dndB-E constitute an operon, as revealed by RT-PCR analysis. An efficient mutation-integration-complementation system was developed to allow for detailed functional analysis of these dnd genes. The Dnd- phenotype caused by specific in-frame deletion of the dndA, C, D, and E genes or the enhanced Dnd phenotype resulting from in-frame deletion of dndB could be restored by expression vectors carrying the corresponding dnd genes. Interestingly, overdosage of DndC or DndD, but not other Dnd proteins, in vivo was found to be detrimental to cell viability. DNA phosphorothioation is a multi-enzymatic and highly coordinated process controlled by five dnd genes. Overexpression of some proteins in vivo prevented growth of host strain, suggesting that expression of the gene cluster is strictly regulated in the native host.
    BMC Microbiology 03/2009; 9:41. · 2.98 Impact Factor
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    ABSTRACT: Tailoring steps are often important for the activity of mature antibiotics. Here, we report that novel decarboxylated FR-008/candicidin derivatives were obtained from the P450 monooxygenase gene fscP mutant of Streptomyces sp. strain FR-008. The toxicity of decarboxylated FR-008/candicidin derivatives has been shown to be greatly reduced compared to that of wild-type FR-008/candicidin.
    Applied and Environmental Microbiology 02/2009; 75(6):1778-81. · 3.95 Impact Factor
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    ABSTRACT: Modifications of the canonical structures of DNA and RNA play critical roles in cell physiology, DNA replication, transcription and translation in all organisms. We now report that bacterial dnd gene clusters incorporate sulfur into the DNA backbone as a sequence-selective, stereospecific phosphorothioate modification. To our knowledge, unlike any other DNA or RNA modification systems, DNA phosphorothioation by dnd gene clusters is the first physiological modification described on the DNA backbone.
    Nature Chemical Biology 12/2007; 3(11):709-10. · 12.95 Impact Factor
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    ABSTRACT: A novel DNA modification system by sulfur (S) in Streptomyces lividans 66 was reported to be encoded by a cluster of five genes designated dndA-E [Zhou, X., He, X., Liang, J., Li, A., Xu, T., Kieser, T., Helmann, J. D., and Deng, Z. (2005) Mol. Microbiol. 57, 1428-1438]. The dndA gene was cloned and the protein product expressed in Escherichia coli, purified to homogeneity, and characterized as a homodimeric protein of ca. 91 kDa. Purified DndA has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate-containing enzyme and was proven to be a cysteine desulfurase able to catalyze removal of elemental S atoms from l-cysteine to produce l-alanine with substrate specificity similar to that of E. coli IscS. DndC was also purified to homogeneity and found to contain a 4Fe-4S cluster by spectral analysis and have obvious ATP pyrophosphatase activity. DndA could catalyze iron-sulfur cluster assembly by activation of apo-Fe DndC protein prepared by removal of its iron-sulfur cluster using alpha,alpha'-dipyridyl. A mutated DndA, with serine substituted for cysteine at position 327, which was confirmed to have lost its corresponding cysteine desulfurase activity, also lost its ability to reactivate the apo-Fe DndC. The likely involvement of an interaction between DndA and DndC in the biochemical pathway for the unusual site-specific DNA modification in S. lividans 66 is discussed.
    Biochemistry 06/2007; 46(20):6126-33. · 3.38 Impact Factor
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    ABSTRACT: In the arsenic resistance gene cluster from the large linear plasmid pHZ227, two novel genes, arsO (for a putative flavin-binding monooxygenase) and arsT (for a putative thioredoxin reductase), were coactivated and cotranscribed with arsR1-arsB and arsC, respectively. Deletion of the ars gene cluster on pHZ227 in Streptomyces sp. strain FR-008 resulted in sensitivity to arsenic, and heterologous expression of the ars gene cluster in the arsenic-sensitive Streptomyces strains conferred resistance on the new hosts. The pHZ227 ArsB protein showed homology to the yeast arsenite transporter Acr3p. The pHZ227 ArsC appears to be a bacterial thioredoxin-dependent ArsC-type arsenate reductase with four conserved cysteine thioredoxin-requiring motifs.
    Applied and Environmental Microbiology 06/2006; 72(5):3738-42. · 3.95 Impact Factor

Publication Stats

150 Citations
68.44 Total Impact Points

Institutions

  • 2014
    • Wuhan University
      Wu-han-shih, Hubei, China
  • 2009–2011
    • Massachusetts Institute of Technology
      • Department of Biological Engineering
      Cambridge, MA, United States
  • 2006–2010
    • Shanghai Jiao Tong University
      • School of Life Science and Biotechnology
      Shanghai, Shanghai Shi, China
    • Huazhong Agricultural University
      Wu-han-shih, Hubei, China
  • 2007–2009
    • Shanghai University
      Shanghai, Shanghai Shi, China