title: A mutant of uracil DNA glycosylase that distinguishes between cytosine and 5-methylcytosine.
abstract: 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.
authors.affiliation: Centre for Biological Sciences, University of Southampton, Highfield, Southampton, United Kingdom.
authors.forename: Scott T
authors.initials: ST
authors.lastname: Kimber
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford, United Kingdom.
authors.forename: Tom
authors.initials: T
authors.lastname: Brown
authors.affiliation: Centre for Biological Sciences, University of Southampton, Highfield, Southampton, United Kingdom.
authors.forename: Keith R
authors.initials: KR
authors.lastname: Fox
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003596:Cytosine / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013941:Thymine / Q000737:chemistry / Q000378:metabolism; D014498:Uracil / Q000737:chemistry / Q000378:metabolism; D051981:Uracil-DNA Glycosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; D014498:Uracil; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D051981:Uracil-DNA Glycosidase; D013941:Thymine
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