title: Fidelity index determination of DNA methyltransferases.
abstract: DNA methylation is the most frequent form of epigenetic modification in the cell, which involves gene regulation in eukaryotes and protection against restriction enzymes in prokaryotes. Even though many methyltransferases exclusively modify their cognate sites, there have been reports of those that exhibit promiscuity. Previous experimental approaches used to characterize these methyltransferases do not provide the exact concentration at which off-target methylation occurs. Here, we present the first reported fidelity index (FI) for a number of DNA methyltransferases. We define the FI as the ratio of the highest amount of methyltransferase that exhibits no star activity (off-target effects) to the lowest amount that exhibits complete modification of the cognate site. Of the methyltransferases assayed, M.MspI and M.AluI exhibited the highest fidelity of ≥250 and ≥500, respectively, and do not show star activity even at very high concentrations. In contrast, M.HaeIII, M.EcoKDam and M.BamHI have the lowest fidelity of 4, 4 and 2, respectively, and exhibit star activity at concentrations close to complete methylation of the cognate site. The fidelity indexes provide vital information on the usage of methyltransferases and are especially important in applications where site specific methylation is required.
authors.affiliation: New England Biolabs Inc., Ipswich, Massachusetts, United States of America.
authors.forename: Janine G
authors.initials: JG
authors.lastname: Borgaro
authors.forename: Nicole
authors.initials: N
authors.lastname: Benner
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Zhu
mesh_terms: D001483:Base Sequence; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000737:chemistry; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000502:physiology; D057075:Enzyme Assays; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000502:physiology; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C056438:DNA modification methylase EcoK; C020098:DNA modification methylase EcoRI; C029641:DNA modification methylase HpaII; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; C027823:Cytosine 5-methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)