Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes

Xu, Shuang-yong; Klein, Pernelle; Degtyarev, S. Kh.; Roberts, Richard J.

doi:10.1038/srep28579

Cited by 11 publications

(30 citation statements)

References 27 publications

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“…MDE digests of substituted phages. For these enzymes, the WebLogos (Fig 5) are compatible with but more relaxed than the site G m5 CNG m5 C proposed for the BisI family by Xu et al [8]. Importantly, most of the information determining cleavage is contained within the 5-base window surrounding the ligation site.…”

Section: Resultsmentioning

confidence: 71%

“…Here we compared EcoBLMcrX with the recently-characterized MDEs [8] NhoI and BceYI. With m5 C G -modified DNA (upper panel), NhoI is distinct from the other two, with little activity.…”

Section: Resultsmentioning

confidence: 99%

“…At least 15 families of MDE have been discovered and characterized [6–8]. Some are used for study of base modification in eukaryotes (e.g.…”

Section: Introductionmentioning

confidence: 99%

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EcoBLMcrX, a classical modification-dependent restriction enzyme in Escherichia coli B: Characterization in vivo and in vitro with a new approach to cleavage site determination

et al. 2017

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Here we characterize the modification-dependent restriction enzyme (MDE) EcoBLMcrX in vivo, in vitro and in its genomic environment. MDE cleavage of modified DNAs protects prokaryote populations from lethal infection by bacteriophage with highly modified DNA, and also stabilizes lineages by reducing gene import when sparse modification occurs in the wrong context. The function and distribution of MDE families are thus important. Here we describe the properties of EcoBLMcrX, an enzyme of the E. coli B lineage, in vivo and in vitro. Restriction in vivo and the genome location of its gene, ecoBLmcrX, were determined during construction and sequencing of a B/K-12 hybrid, ER2566. In classical restriction literature, this B system was named r6 or rglAB. Like many genome defense functions, ecoBLmcrX is found within a genomic island, where gene content is variable among natural E. coli isolates. In vitro, EcoBLMcrX was compared with two related enzymes, BceYI and NhoI. All three degrade fully cytosine-modified phage DNA, as expected for EcoBLMcrX from classical T4 genetic data. A new method of characterizing MDE specificity was developed to better understand action on fully-modified targets such as the phage that provide major evolutionary pressure for MDE maintenance. These enzymes also cleave plasmids with m5C in particular motifs, consistent with a role in lineage-stabilization. The recognition sites were characterized using a site-ranking approach that allows visualization of preferred cleavage sites when fully-modified substrates are digested. A technical constraint on the method is that ligation of one-nucleotide 5' extensions favors G:C over A:T approximately five-fold. Taking this bias into account, we conclude that EcoBLMcrX can cleave 3' to the modified base in the motif Rm5C|. This is compatible with, but less specific than, the site reported by others. Highly-modified site contexts, such as those found in base-substituted virulent phages, are strongly preferred.

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Section: Resultsmentioning

confidence: 71%

“…Here we compared EcoBLMcrX with the recently-characterized MDEs [8] NhoI and BceYI. With m5 C G -modified DNA (upper panel), NhoI is distinct from the other two, with little activity.…”

Section: Resultsmentioning

confidence: 99%

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EcoBLMcrX, a classical modification-dependent restriction enzyme in Escherichia coli B: Characterization in vivo and in vitro with a new approach to cleavage site determination

et al. 2017

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“…In the restriction enzyme data base (REBASE)(www.rebase.neb.com) there are 517 characterized and putative Type IIM REases from bacteria 34 . Most of them belong to the BisI family 37 , MspJI family 38 , DpnI family 39 , and PvuRts1I family 20 of enzymes. Some BisI homologs with low sequence similarities have diverged into new modification-dependent endonuclease specificities (SYX, unpublished results).…”

Section: Discussionmentioning

confidence: 99%

Restriction and modification of deoxyarchaeosine (dG+)-containing phage 9 g DNA

Tsai

Corrêa

et al. 2017

Sci Rep

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E. coli phage 9 g contains the modified base deoxyarchaeosine (dG+) in its genome. The phage encodes its own primase, DNA ligase, DNA polymerase, and enzymes necessary to synthesize and incorporate dG+. Here we report phage 9 g DNA sensitivity to >200 Type II restriction endonucleases (REases). Among the REases tested approximately 29% generated complete or partial digestions, while the remaining 71% displayed resistance to restriction. Phage 9 g restriction fragments can be degraded by DNA exonucleases or ligated by T3 and T4 DNA ligases. In addition, we examined a number of cytosine and adenine methyltransferases to generate double base modifications. M.AluI, M.CviPI, M.HhaI, and M.EcoGII were able to introduce 5mC or N6mA into 9 g DNA as confirmed by partial resistance to restriction and by liquid chromatography-mass spectrometry. A number of wild-type E. coli bacteria restricted phage 9 g, indicating natural restriction barriers exist in some strains. A BlastP search of GenBank sequences revealed five glutamine amidotransferase-QueC homologs in Enterobacteria and Pseudomonas phage, and distant homologs in other phage and bacterial genomes, suggesting that dG+ is not a rare modification. We also mapped phage 9 g DNA packaging (pac) site containing two 21-bp direct repeats and a major terminase cleavage site in the phage genome.

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“…In addition, 6m A is known to play a role in phage growth limitation (Pgl) systems, but the hypothesized 6m A-dependent REase has not yet been identified ( 13 ). Some cytosine modification dependent REases cleave highly GC-rich target sequences containing several modified cytosines directly within the recognition sequence (GlaI ( 14 ), BisI ( 15 ), Eco15I ( 15 ) and EcoBLMcrX ( 16 )). However, more typically, cytosine modification dependent REases cleave at considerable distance from one or more modified cytosine bases.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of the modification-dependent SRA-HNH endonuclease TagI

Kisiala

Copelas

Czapinska

et al. 2018

Nucleic Acids Research

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TagI belongs to the recently characterized SRA-HNH family of modification-dependent restriction endonucleases (REases) that also includes ScoA3IV (Sco5333) and TbiR51I (Tbis1). Here, we present a crystal structure of dimeric TagI, which exhibits a DNA binding site formed jointly by the nuclease domains, and separate binding sites for modified DNA bases in the two protomers. The nuclease domains have characteristic features of HNH/ββα-Me REases, and catalyze nicks or double strand breaks, with preference for /RY and RYN/RY sites, respectively. The SRA domains have the canonical fold. Their pockets for the flipped bases are spacious enough to accommodate 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC), but not glucosyl-5-hydroxymethylcytosine (g5hmC). Such preference is in agreement with the biochemical determination of the TagI modification dependence and the results of phage restriction assays. The ability of TagI to digest plasmids methylated by Dcm (C5mCWGG), M.Fnu4HI (G5mCNGC) or M.HpyCH4IV (A5mCGT) suggests that the SRA domains of the enzyme are tolerant to different sequence contexts of the modified base.

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Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes

Cited by 11 publications

References 27 publications

EcoBLMcrX, a classical modification-dependent restriction enzyme in Escherichia coli B: Characterization in vivo and in vitro with a new approach to cleavage site determination

EcoBLMcrX, a classical modification-dependent restriction enzyme in Escherichia coli B: Characterization in vivo and in vitro with a new approach to cleavage site determination

Restriction and modification of deoxyarchaeosine (dG+)-containing phage 9 g DNA

Crystal structure of the modification-dependent SRA-HNH endonuclease TagI

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