DNA phosphorothioate modifications influence the global transcriptional response and protect DNA from double-stranded breaks

Wang

Proc. Natl. Acad. Sci. U.S.A.

et al. 2017

Self Cite

Explosive growth in the study of microbial epigenetics has revealed a diversity of chemical structures and biological functions of DNA modifications in restriction-modification (R-M) and basic genetic processes. Here, we describe the discovery of shared consensus sequences for two seemingly unrelated DNA modification systems, 6m A methylation and phosphorothioation (PT), in which sulfur replaces a nonbridging oxygen in the DNA backbone. Mass spectrometric analysis of DNA from Escherichia coli B7A and Salmonella enterica serovar Cerro 87, strains possessing PT-based R-M genes, revealed d(G PS 6m A) dinucleotides in the G PS 6m AAC consensus representing ∼5% of the 1,100 to 1,300 PT-modified d(G PS A) motifs per genome, with 6m A arising from a yet-to-be-identified methyltransferase. To further explore PT and 6m A in another consensus sequence, G PS 6m ATC, we engineered a strain of E. coli HST04 to express Dnd genes from Hahella chejuensis KCTC2396 (PT in G PS ATC) and Dam methyltransferase from E. coli DH10B ( 6m A in G 6m ATC). Based on this model, in vitro studies revealed reduced Dam activity in G PS ATC-containing oligonucleotides whereas single-molecule real-time sequencing of HST04 DNA revealed 6m A in all 2,058 G PS ATC sites (5% of 37,698 total GATC sites). This model system also revealed temperature-sensitive restriction by DndFGH in KCTC2396 and B7A, which was exploited to discover that 6m A can substitute for PT to confer resistance to restriction by the DndFGH system. These results point to complex but unappreciated interactions between DNA modification systems and raise the possibility of coevolution of interacting systems to facilitate the function of each.T he emergence of convergent technologies has led to a growing appreciation for the diversity of DNA modifications in microbial epigenetics and restriction-modification (R-M) systems (1-3). DNA methylation, the most extensively studied genetic modification, was originally discovered in bacteria in the context of R-M systems involving a methyltransferase (MTase) that modifies "self" DNA at specific target sites and a cognate restriction endonuclease (REase) that discriminates and destroys unmodified invading DNA (3-5). R-M systems are ubiquitous in prokaryotes and are classified into four types (I, II, III, and IV) based on their molecular structure, sequence recognition, cleavage position, and cofactor requirements (3, 6, 7). However, some MTases exist alone, without an apparent cognate REase partner. These so-called "orphan" MTases include DNA adenine methylase (Dam), which modifies the adenine N-6 in the GATC motif, DNA cytosine methylase (Dcm), which methylates C-5 of the second cytosine in CC(A/T) GG sequences, and cell cycle-regulated methylase (CcrM), which methylates the N-6 of adenine in GANTC (N = A, T, C, or G) (8). Despite the absence of cognate REases, orphan MTases still confer immunity against the virulence of a parasitic R-M complex with the same target sites (9). In addition to defense against bacteriophages and transposons, DNA m...

Section: Development Of a Temperature-dependent Pt-associated Restricmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Convergence of DNA methylation and phosphorothioation epigenetics in bacterial genomes

Wang

Proc. Natl. Acad. Sci. U.S.A.

et al. 2017

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Medicinal Research Reviews

“…Current data suggest that DNA phosphorothioate modifications are part of a restriction‐modification (R‐M) system, with similarities to methylation‐based R‐M systems, in which DndABCDE functions as the M component and confers the DNA with phosphorothioate modification in a sequence‐specific manner, whereas DndFGH functions as the R component to recognize and hydrolyze non‐phosphorothioate‐protected invading DNA . The loss of phosphorothioate protection in the host results in DNA damage and triggers the cellular SOS response …”

Section: Regulation Of Gene Cluster Expressionmentioning

confidence: 99%

Genome Engineering and Modification Toward Synthetic Biology for the Production of Antibiotics

Zou

Wang

et al. 2017

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Antibiotic production is often governed by large gene clusters composed of genes related to antibiotic scaffold synthesis, tailoring, regulation, and resistance. With the expansion of genome sequencing, a considerable number of antibiotic gene clusters has been isolated and characterized. The emerging genome engineering techniques make it possible towards more efficient engineering of antibiotics. In addition to genomic editing, multiple synthetic biology approaches have been developed for the exploration and improvement of antibiotic natural products. Here, we review the progress in the development of these genome editing techniques used to engineer new antibiotics, focusing on three aspects of genome engineering: direct cloning of large genomic fragments, genome engineering of gene clusters, and regulation of gene cluster expression. This review will not only summarize the current uses of genomic engineering techniques for cloning and assembly of antibiotic gene clusters or for altering antibiotic synthetic pathways but will also provide perspectives on the future directions of rebuilding biological systems for the design of novel antibiotics.

“…(C) The relative positions of residues G21, G24 and L58 in each monomer. Once phosphorothioate modification is abolished, Salmonella enterica suffers dsDNA damage from the unrestrained restriction activity of DndFGH, which consequently leads to growth defects and triggers the SOS response [20,21]. The tetramer conformation is drawn in an orientation similar to that in the left of (A).…”

mentioning

confidence: 99%

“…DndE monomers are shown in pink, cyan, green and yellow cartoon modes, respectively. DndFGH proteins function as the R component to recognize and destroy nonphosphorothioate modified foreign DNA in an mechanism that remains unclear [20,22]. (D) The mutations on the residues G21, G24 and L58 do not affect the dnd phenotype.…”

mentioning

confidence: 99%

Investigating the interactions between DNA and DndE during DNA phosphorothioation

et al. 2019

The DNA phosphorothioate modification is a novel physiological variation in bacteria. DndE controls this modification by binding to dsDNA via a mechanism that remains unclear. Structural analysis of the wild‐type DndE tetramer suggests that a positively charged region in its center is important for DNA binding. In the present study, we replaced residues G21 and G24 in this region with lysines, which increases the DNA binding affinity but does not affect the DNA degradation phenotype. Structural analysis of the mutant indicates that it forms a new tetrameric conformation and that DndE interacts with DNA as a monomer rather than as a tetramer. A structural model of the DndE–DNA complex, based on its structural homolog P22 Arc repressor, indicates that two flexible loops in DndE are determinants of DNA binding