ArticleNucleic acids research2014
Engineering nicking enzymes that preferentially nick 5-methylcytosine-modified DNA.
Article in Nucleic acids research, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
What it found
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
6 citing papers in PubMed, 11 citations in OpenAlex.
- Positioning of nucleosomes containing γ-H2AX precedes active DNA demethylation and transcription initiation.Nature communications · 2021Article
- Activity and structure of EcoKMcrA.Nucleic acids research · 2018Article
- Structural and functional characterization of deep-sea thermophilic bacteriophage GVE2 HNH endonuclease.Scientific reports · 2017Article
- Complete Genome Sequence Analysis of Bacillus subtilis T30.Genome announcements · 2015Article
- Article
- Type II restriction endonucleases--a historical perspective and more.Nucleic acids research · 2014Review
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
N.ϕGamma is a strand-specific and site-specific DNA nicking enzyme (YCG↓GT or AC↑CGR). Here we describe the isolation of single and double mutants of N.ϕGamma with attenuated activity. The nicking domains (NDs) of E59A and 11 double mutants were fused to the 5mCG-binding domain of MBD2 and generated fusion enzymes that preferentially nick 5mCG-modified DNA. The CG dinucleotide can be modified by C5 methyltransferases (MTases) such as M.SssI, M.HhaI or M.HpaII to create composite sites AC↑YGG N(8-15) 5mCG. We also constructed a fusion enzyme 2xMBD2-ND(N.BceSVIII) targeting more frequent composite sites AS↑YS N(5-12) 5mCG in Mn2+ buffer. 5mCG-dependent nicking requires special digestion conditions in high salt (0.3 M KCl) or in Ni2+ buffer. The fusion enzyme can be used to nick and label 5mCG-modified plasmid and genomic DNAs with fluorescently labeled Cy3-dUTP and potentially be useful for diagnostic applications, DNA sequencing and optical mapping of epigenetic markers. The importance of the predicted catalytic residues D89, H90, N106 and H115 in N.ϕGamma was confirmed by mutagenesis. We found that the wild-type enzyme N.ϕGamma prefers to nick 5mCG-modified DNA in Ni2+ buffer even though the nicking activity is sub-optimal compared to the activity in Mg2+ buffer.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.