Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing

Clark, Tyson A.; Murray, Iain A.; Morgan, Richard D.; Kislyuk, Andrey; Spittle, Kristi; Boitano, Matthew; Fomenkov, Alexey; Roberts, Richard J.; Korlach, Jonas

doi:10.1093/nar/gkr1146

Cited by 297 publications

(317 citation statements)

References 37 publications

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“…However, contrary to those studies, recent data from single-molecule, real-time (SMRT) DNA sequencing of bacterial genomes showed that many of the MTases tested exhibited high fidelity (218). Interestingly, that study also revealed that the E. coli Dam MTase is highly promiscuous (218).…”

Section: Future Perspectivesmentioning

confidence: 75%

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Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

Vasu

Nagaraja

2013

Microbiol Mol Biol Rev

523

462

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SUMMARY Restriction-modification (R-M) systems are ubiquitous and are often considered primitive immune systems in bacteria. Their diversity and prevalence across the prokaryotic kingdom are an indication of their success as a defense mechanism against invading genomes. However, their cellular defense function does not adequately explain the basis for their immaculate specificity in sequence recognition and nonuniform distribution, ranging from none to too many, in diverse species. The present review deals with new developments which provide insights into the roles of these enzymes in other aspects of cellular function. In this review, emphasis is placed on novel hypotheses and various findings that have not yet been dealt with in a critical review. Emerging studies indicate their role in various cellular processes other than host defense, virulence, and even controlling the rate of evolution of the organism. We also discuss how R-M systems could have successfully evolved and be involved in additional cellular portfolios, thereby increasing the relative fitness of their hosts in the population.

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Section: Future Perspectivesmentioning

confidence: 75%

“…Interestingly, that study also revealed that the E. coli Dam MTase is highly promiscuous (218). Thus, those enzymes which have a broad specificity could have evolved to serve other roles in the cell, viz., the regulation of transcription and the modulation of DNA transaction processes.…”

Section: Future Perspectivesmentioning

confidence: 94%

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

Vasu

Nagaraja

2013

Microbiol Mol Biol Rev

523

462

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“…In an effort to identify all three oxi-mC species simultaneously in the C. cinerea DNA, we turned to SMRT sequencing (29)(30)(31). 5mC and 5hmC are only weakly detected by SMRT sequencing (15), whereas 5caC yields a strong signal (16).…”

Section: Resultsmentioning

confidence: 99%

Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea

Chávez

Huang

Luong

et al. 2014

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

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TET/JBP enzymes oxidize 5-methylpyrimidines in DNA. In mammals, the oxidized methylcytosines (oxi-mCs) function as epigenetic marks and likely intermediates in DNA demethylation. Here we present a method based on diglucosylation of 5-hydroxymethylcytosine (5hmC) to simultaneously map 5hmC, 5-formylcytosine, and 5-carboxylcytosine at near-base-pair resolution. We have used the method to map the distribution of oxi-mC across the genome of Coprinopsis cinerea, a basidiomycete that encodes 47 TET/JBP paralogs in a previously unidentified class of DNA transposons. Like 5-methylcytosine residues from which they are derived, oxi-mC modifications are enriched at centromeres, TET/JBP transposons, and multicopy paralogous genes that are not expressed, but rarely mark genes whose expression changes between two developmental stages. Our study provides evidence for the emergence of an epigenetic regulatory system through recruitment of selfish elements in a eukaryotic lineage, and describes a method to map all three different species of oxi-mCs simultaneously.T he discovery that oxidative modifications of DNA bases are catalyzed by the TET/JBP family of 2-oxoglutarate and irondependent dioxygenases (1-4) opened a major area of research into the epigenetics of various eukaryotic lineages (reviewed in refs. 5-7). In metazoans, the TET/JBP family is represented by TET proteins, which are present in all animals that are known to possess DNA cytosine methylation (3,8,9). The three TET paralogs of vertebrates have been shown to catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) (2), which is progressively oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (10-12). The discovery of these oxidized methylcytosine (oxi-mC) modifications triggered a flurry of studies on the mammalian TET paralogs; the reports increasingly point toward important roles for these oxidative modifications as intermediates in the enigmatic process of DNA demethylation, and also as epigenetic marks in their own right (13, 14) (reviewed in refs. 5-7). TET proteins have roles in diverse biological processes, including epigenetic regulation of gene transcription, embryonic development, stem cell function, and cancer (5), but the mechanisms underlying their biological activities are still poorly defined.Several methods have been developed to profile individual oxi-mC species at base resolution in genomic DNA (reviewed in ref. 5). However, none of these methods can simultaneously map all three oxi-mC species-5hmC, 5fC, and 5caC-at the same time; rather, they rely on chemical or enzymatic conversion of individual oxi-mCs followed by bisulfite sequencing. Two recent sequencing technologies, single-molecule, real-time (SMRT) sequencing and protein nanopore sequencing, are capable of recognizing modified bases in unamplified genomic DNA (15-18). In SMRT sequencing, 5mC and 5hmC are barely detectable in unmodified DNA, whereas 5fC and 5caC yield a robust kinetic signature (16). As 5hmC is an abundant oxi-mC modification in ...

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“…SMRTbell template libraries were prepared as previously described (53,54). SMRT sequencing was carried out on the PacBio RSII instrument using standard protocols for large insert SMRTbell libraries.…”

Section: Methodsmentioning

confidence: 99%

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk

O’Connor

Fung

Sharp

et al. 2014

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

130

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Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea, wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.Teredinidae | endosymbionts | symbiosis | xylotrophy | carbohydrate-active enzymes

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Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing

Cited by 297 publications

References 37 publications

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk

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