Engineering nicking enzymes that preferentially nick 5-methylcytosine-modified DNA

N.phi Gamma is a strand-specific and site-specific DNA nicking enzyme (YCG down arrow GT or AC up arrow CGR). Here we describe the isolation of single and double mutants of N.phi Gamma 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 ACa up arrow YGG N(8-15) 5mCG. We also constructed a fusion enzyme 2xMBD2-ND(N.BceSVIII) targeting more frequent composite sites AS up arrow 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.phi Gamma was confirmed by mutagenesis. We found that the wild-type enzyme N.phi Gamma prefers to nick 5mCG-modified DNA in Ni2+ buffer even though the nicking activity is sub-optimal compared to the activity in Mg2+ buffer.