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.
© 2014 The Author(s). Published by Oxford University Press [on behalf of Nucleic Acids Research].
Nicking activity assay for mutants of N.ϕGamma. (
A) One microgram of pBR322 was digested by purified enzyme (small scale) in a Mg 2+ buffer (NEB buffer 2). L = 2-log DNA ladder (NEB), WT = wild-type N.ϕGamma. Single mutant aa substitution was indicated on top of each lane. NC: nicked circular; L: linear; SC: supercoiled. ( B) Run-off sequencing of plasmid DNA nicked by the full-length E59A nickase. Arrows indicate nicked site with double peaks at A/C. ( C) Schematic diagram of DNA run-off sequencing to determine the nicked site. A doublet peak with extra A indicates the nicked site. The height of base-call peaks following the doublet drops off due to the nicked template.
Schematic diagram of 2xMBD2-ND(N.ϕGamma) functional domains and fusion enzyme purification. (
A) A schematic diagram of the fusion protein 2xMBD2-ND(N.ϕGamma). 5mCG BD, 5mCG-binding domain; 2× = duplicated 5mCG BD; ND, nicking domain. ( B) SDS-PAGE analysis of partially purified fusion protein. The molecular mass of the fusion protein is predicted to be ∼36 kDa. Lane 2, fusion protein purified from a chitin column after DTT cleavage. Lanes 3–9, eluted fractions from a heparin Sepharose column.
Effect of KCl concentration on the nicking specificity of 2xMBD2-ND(E59A) fusion enzyme. (
A) Nicking reaction carried out in Mg 2+ buffer supplemented with 0.1–0.3 M KCl. 0.5 μg of pBR322 (unmodified) was digested by 2.5 μg of the fusion enzyme in NEB buffer 2 (50 mM NaCl, 10 mM MgCl 2, 10 mM Tris-HCl, pH 7.8) plus different concentrations of KCl for 1 h at 37°C. NC: nicked circular; L: linear; SC: supercoiled. ( B) Same digestion condition as in (A), except that 5mCG-modified pBR322 was used. ( C) Nicking reaction carried out in NEB buffer 2 plus 0.3 M KCl with partially purified E59A and seven nicking domains of double mutants (in E59A background) with modified (M +) and unmodified (M −) pBR322. ( D) Characterization of the nicking site of 2xMBD2-ND(E59A) in Mg 2+ buffer. Run-off sequencing of modified and nicked pBR322. One of the composite nicking sites is (CG N 6–14) AC↑CGG N 13 CG. The arrow indicates the nicking site with A/C doublet. Both strands were sequenced. ( E) A time course of digestion in 10–60 min with fixed amount of fusion enzyme and DNA.
Nicking activity assay for 2xMBD2-ND(E59A) fusion enzyme for methylated DNA in Mn
2+ buffer. ( A) 0.5 μg of 5mCG-modified pBR322 was digested by different amounts of enzyme (0.3–1.3 μg) in a buffer containing 10 mM MnCl 2, 10 mM Tris-HCl, pH 7.8, 50 mM NCl and 0.3 M KCl in 30 min, 45 min and 1 h at 37°C. M+, 5mCG-modified substrate; M−, unmodified substrate. ( B) Run-off sequencing of the pre-nicked modified DNA. The composite nicking site is (CGN 6–17) AC↑CC N 11 CG. Arrow indicates the nicked site with A/C doublet.
Nicking activity assay of 2xMBD2-ND(E59A) using a pUC19 derivative. (
A) One microgram of M.HhaI-modified or unmodified pUC19 was digested by 2.5 μg of the fusion nicking enzyme in NEB buffer 2 supplemented with 0.3 M KCl for 2 h at 37°C. M.HhaI modifies the site GCGC to produce G 5mCGC, which subsequently serves as the substrate for the 5mCG-binding domain. NC: nicked circular; SC: supercoiled. ( B) Run-off sequencing of nicked DNA modified by M.HhaI. The composite site ACTGG N 14 G CGC is shown. Arrow indicates the nicking site with A/T doublet. No nick was found in sequencing of the opposite strand (top panel).
Nicking activity assay in buffers containing different metal ions for the fusion enzymes 2xMBD2-ND(E59A/R62A) and 2xMBD2-ND(E59A/T119A) (nicking domain double mutants). Methylated pBR322 (0.5 μg) by M.SssI (M) or unmodified (U) were digested by 2.5 μg of the fusion enzyme in buffers containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl and supplemented with different metal ions for 1 h at 37°C. The metal ions and concentration were indicated on top of each lane. Nicking activity was detected in Mg
2+, Mn 2+ and Ni 2+ buffers for 2xMBD2-ND(E59A/R62A); nicking activity was detected in Mn 2+ and Ni 2+ buffers for 2xMBD2-ND(E59A/T119A). These digestions did not include 0.3 M KCl.
Purification of 2xMBD2-ND(N.BceSVIII) fusion enzyme and nicking activity assay. (
A) SDS-PAGE analysis of purified fusion enzyme 2xMBD2-ND(N.BceSVIII). The molecular mass of the fusion enzyme is predicted to be ∼34.6 kDa. ( B) Methylated pBR322 DNA (0.5 μg) by M.SssI (M) or unmodified (U) were digested by 2.5 μg of the fusion enzyme in buffers containing 10 mM Tris-HCl, pH 7.8, 50 mM of NaCl supplemented with 10 mM Mn 2+ or 10 mM Mg 2+ and 0.1–0.3 M KCl for 1 h at 37°C. NC: nicked circular; L: linear; SC: supercoiled; U: unmodified DNA; M: methylated DNA. ( C) Run-off sequencing of modified and nicked DNA in Mn 2+ buffer with 0.3 M KCl. The composite site (CGN 1) ACCC N (5–17) CG is shown. Five CG sites are present in this region.
Digestion 5mCG-modified and unmodified duplex oligos in Ni
2+ buffer by the fusion enzyme 2xMBD2-ND(E59A/R62A), or in Mg 2+ and Ni 2+ buffers by the WT nicking enzyme N.ϕGamma (full-length). ( A) Digestion of M + oligos by the fusion enzyme 2xMBD2-ND(E59A/R62A) in Ni 2 buffer, by WT N.ϕGamma in Ni 2+ or Mg 2+ buffer, and by HpaII restriction enzyme in buffer 4, respectively. Arrow indicates nicked product and dashed arrow indicates dsDNA cleavage product. ( B) Digestion of unmodified (M ) oligos by the fusion enzyme 2xMBD2-ND(E59A/R62A) in Ni − 2+ buffer, by WT N.ϕGamma in Ni 2+ or Mg 2+ buffer, and by HpaII restriction enzyme in buffer 4, respectively. ( C) Digestion of M + and M oligos by the WT enzyme N.ϕGamma (in 2-fold serial dilution) in Ni − 2+ buffer at 37°C for 30 min. ( D) Digestion of M + oligos by the fusion enzyme in 2xMBD2-ND(E59A/R62A) in 2-fold serial dilution in Ni 2+ buffer at 37°C for 30 min. ( E) Digestion of M + oligos by the fusion enzyme in 2xMBD2-ND(E59A/R62A) in Ni 2+ buffer (0.1–5 mM) at 37°C for 30 min. Lane 7 digestion included 1 mM Ni 2+ and 10 mM EDTA. Lane 10, unmodified oligos cleaved by HpaII restriction enzyme.
Labeling of nicked plasmid and genomic DNAs by strand-displacement DNA synthesis. (
A) One microgram of pBR322 methylated by M.SssI (M+) or unmodified (M−) was nicked by 2.5 μg of 2xMBD2-ND(E59A) in Mg 2+ buffer containing 0.25 M KCl for 1 h at 37°C. The labeling was performed through 5 min incubation at 25°C with 10 units of Klenow fragment of E. coli DNA polymerase I and a dNTP mix containing Cy3-dUTP. ( B) NIH 3T3 mouse genomic DNA was nicked by 2.5 μg of 2xMBD2-ND(E59A) in a Mg 2+ buffer containing 0.3 M KCl for 1 h at 37°C. DNA labeling was performed through 10 min incubation at 25°C with 10 units of Klenow fragment of E. coli DNA polymerase I and a dNTP mix containing Cy3-dUTP. The control enzyme Nt.BspQI nicks the DNA sites GCTCTTCN↓N.
All figures (9)
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