A Unique m6A-Dependent Restriction Endonuclease from an Archaeal Virus

Lu, Xin; Huang, Fengtao; Cheng, Rui; Zhu, Bin

doi:10.1128/spectrum.04262-22

Cited by 4 publications

(8 citation statements)

References 35 publications

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“…Some known wH domains are adenine methylation (6mA)-specific. This notion was originally suggested by the demonstration of 6mA specificity of the DpnI wH domain and further strengthened by the observation of adenine specificity of the wH domain of HHPV4I (Lu et al, 2023), which was reported when this study was being finalized. In the hope of finding new adenine methylation-specific endonucleases, we searched for fusions of wH domains with endonuclease domains known to play roles in R-M (Pingoud and Jeltsch, 2001).…”

Section: Bioinformatic Screen Of Wh Domain Endonucleasesmentioning

confidence: 54%

“…6× His-tagged HhiV4I (see Supplementary Figure S5 ) was subjected to three-step chromatography (Ni-agarose column, DEAE column, and Heparin agarose column). Compared to the recently published paper on the same enzyme ( Lu et al, 2023 ), two additional chromatography steps were used (DEAE and Heparin columns). Unfortunately, the Heparin agarose chromatography step was less efficient for purification than is typical for other DNA-binding proteins because HhiV4I was in the flow-through and did not bind to the Heparin column, as would be expected for a typical nucleic acid-binding protein.…”

Section: Resultsmentioning

confidence: 99%

“…More recently, a winged helix domain was also implicated in the sensing of adenine methylation, also in the G6mATC context. HHPV4I (also called HhiV4I; Lu et al, 2023 ) is a three-domain enzyme, with a PUA (SRA)-like domain at the N-terminus, a winged helix domain in the middle, and an HNH endonuclease domain at the C-terminus. The PUA superfamily domain, described as an SRA domain by Lu et al, appears not to be involved in DNA modification sensing ( Lu et al, 2023 ).…”

Section: Introductionmentioning

confidence: 99%

“…HHPV4I (also called HhiV4I; Lu et al, 2023 ) is a three-domain enzyme, with a PUA (SRA)-like domain at the N-terminus, a winged helix domain in the middle, and an HNH endonuclease domain at the C-terminus. The PUA superfamily domain, described as an SRA domain by Lu et al, appears not to be involved in DNA modification sensing ( Lu et al, 2023 ). By contrast, the winged helix domain directed preferential cleavage of Dam + over Dam − DNA, and it has much higher affinity to Dam + than to Dam − DNA in gel shift experiments.…”

Section: Introductionmentioning

confidence: 99%

“…By contrast, the winged helix domain directed preferential cleavage of Dam + over Dam − DNA, and it has much higher affinity to Dam + than to Dam − DNA in gel shift experiments. In contrast to DpnI, HHPV4I (HhiV4I) cleaves at a distance from the site of adenine methylation, suggesting that the endonuclease domain is directed by the winged helix domain and does not sense adenine methylation on its own ( Lu et al, 2023 ).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of winged helix domain fusion endonucleases as N6-methyladenine-dependent type IV restriction systems

Helbrecht,

Heiter,

Yang

et al. 2024

Front. Microbiol.

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Winged helix (wH) domains, also termed winged helix-turn-helix (wHTH) domains, are widespread in all kingdoms of life and have diverse roles. In the context of DNA binding and DNA modification sensing, some eukaryotic wH domains are known as sensors of non-methylated CpG. In contrast, the prokaryotic wH domains in DpnI and HhiV4I act as sensors of adenine methylation in the 6mApT (N6-methyladenine, 6mA, or N6mA) context. DNA-binding modes and interactions with the probed dinucleotide are vastly different in the two cases. Here, we show that the role of the wH domain as a sensor of adenine methylation is widespread in prokaryotes. We present previously uncharacterized examples of PD-(D/E)XK—wH (FcyTI, Psp4BI), PUA—wH—HNH (HtuIII), wH—GIY-YIG (Ahi29725I, Apa233I), and PLD—wH (Aba4572I, CbaI) fusion endonucleases that sense adenine methylation in the Dam+ Gm6ATC sequence contexts. Representatives of the wH domain endonuclease fusion families with the exception of the PLD—wH family could be purified, and an in vitro preference for adenine methylation in the Dam context could be demonstrated. Like most other modification-dependent restriction endonucleases (MDREs, also called type IV restriction systems), the new fusion endonucleases except those in the PD-(D/E)XK—wH family cleave close to but outside the recognition sequence. Taken together, our data illustrate the widespread combinatorial use of prokaryotic wH domains as adenine methylation readers. Other potential 6mA sensors in modified DNA are also discussed.

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Section: Bioinformatic Screen Of Wh Domain Endonucleasesmentioning

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of winged helix domain fusion endonucleases as N6-methyladenine-dependent type IV restriction systems

Helbrecht,

Heiter,

Yang

et al. 2024

Front. Microbiol.

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Prokaryotic winged helix domains as dsDNA adenine methylation sensors

Heiter

Yang

Lutz

et al. 2023

Preprint

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Winged helix (wH) domains, also termed winged helix-turn-helix (wHTH) domains, are widespread in all kingdoms of life, and have diverse roles. In the context of DNA binding and DNA modification sensing, some eukaryotic wH domains are known as sensors of non-methylated CpG. In contrast, the prokaryotic wH domains in DpnI and phi.HhiV4I act as sensors of adenine methylation in 6mApT (6mA = N6mA) context. DNA binding modes and interactions with the probed dinucleotide are vastly different in the two cases. Here, we show that the role of the wH domain as a sensor of adenine methylation is widespread in prokaryotes. We present previously uncharacterized examples of PD-(D/E)XK-wH (FcyTI, Psp4BI), PUA-wH-HNH (HtuIII, Hsa13891I), wH-GIY-YIG (Ahi29725I, Apa233I) and PLD-wH (Aba4572I, CbaI) fusion endonucleases that sense adenine methylation in the Dam G6mATC, and possibly other, slightly more relaxed contexts. Representatives of the wH domain endonuclease fusion families with the exception of the PLD-wH family could be purified, and an in vitro preference for adenine methylation in the Dam context could be demonstrated. Like most other MDREs, the new fusion endonucleases except those in the PD-(D/E)XK-wH family cleave close to, but outside the recognition sequence. Taken together, our data illustrate the widespread combinatorial use of prokaryotic wH domains as adenine methylation sensors.

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Type II restriction of 2-aminoadenine (dZ) modified DNA and production of dZ-modified plasmid inE. coli

Yang,

Kuska,

Dai

et al. 2023

Preprint

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The modified DNA base 2,6 aminopurine (2-aminoadenine, (d)Z base) was originally found in phages to counteract host encoded restriction systems. However, only a limited number of restriction endonucleases (REases) have been tested on dZ-modified DNA. Herein we report the results of 147 REases activity on dZ-modified PCR DNA. Among the enzymes tested, 53.1% are resistant or partially resistant, and 46.9% are sensitive when the restriction sites contain 1 to 6 modified bases. Sites with 4-6 dZ substitutions are most likely resistant to Type II restriction. Our results support the notion that dZ-modified phage genomes are evolved to combat host-encoded restriction systems. dZ-modified DNA can also slow down phage T5 exonuclease degradation, but it has no effect on RecBCD digestion. When two genes for dZ biosynthesis and one gene for dATP hydrolysis from Salmonella phage PMBT28 (purZ (adenylosuccinate synthetase), datZ (dATP triphosphohydrolase), and mazZ ((d)GTP-specific diphosphohydrolase) were cloned into E. coli plasmid, dZ incorporation level reached 19-20% dZ/(dZ+dA). dZ level can be further increased to 28.9-44.3% with co-expression of a DNA polymerase gene from the same phage. High level of dZ incorporation in recombinant plasmid is possible by co-expression of purZ, mazZ, datZ and phage DNA helicase, dpoZ (DNA polymerase) and ssb (single-stranded DNA binding protein SSB). This work has a general interest for molecular biologists working on dZ DNA modification and restriction systems. It provides a foundation for future research on screening dZ-dependent Type IV restriction systems. The results presented herein may have implication in gene therapy utilizing dZ-modified DNA, provided that human RNA polymerase variants can efficiently perform transcription from a dZ-modified template.

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A Unique m6A-Dependent Restriction Endonuclease from an Archaeal Virus

Cited by 4 publications

References 35 publications

Characterization of winged helix domain fusion endonucleases as N6-methyladenine-dependent type IV restriction systems

Characterization of winged helix domain fusion endonucleases as N6-methyladenine-dependent type IV restriction systems

Prokaryotic winged helix domains as dsDNA adenine methylation sensors

Type II restriction of 2-aminoadenine (dZ) modified DNA and production of dZ-modified plasmid inE. coli

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