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We demonstrate that a mutant of uracil DNA glycosylase (N123D:L191A) distinguishes between cytosine and methylcytosine. Uracil DNA glycosylase (UDG) efficiently removes uracil from DNA in a reaction in which the base is flipped into the enzyme's active site. Uracil is selected over cytosine by a pattern of specific hydrogen bonds, and thymine is excluded by steric clash of its 5-methyl group with Y66. The N123D mutation generates an enzyme that excises cytosine. This N123D:L191A mutant excises C when it is mispaired with A or opposite an abasic site, but not when it is paired with G. In contrast no cleavage is observed with any substrates that contain 5-methylcytosine. This enzyme may offer a new approach for discriminating between cytosine and 5-methylcytosine.

Original publication

DOI

10.1371/journal.pone.0095394

Type

Journal article

Journal

PLoS One

Publication Date

2014

Volume

9

Keywords

5-Methylcytosine, Bacterial Proteins, Base Sequence, Cytosine, DNA, Escherichia coli, Gene Expression, Molecular Sequence Data, Mutation, Oligonucleotides, Recombinant Proteins, Substrate Specificity, Thymine, Uracil, Uracil-DNA Glycosidase