A mutant of uracil DNA glycosylase that distinguishes between cytosine and 5-methylcytosine.

• Main Authors: Scott T Kimber, Tom Brown, Keith R Fox
• Format: Article
• Language: English
• Published: Public Library of Science (PLoS) 2014-01-01
• Series: PLoS ONE
• Online Access: http://europepmc.org/articles/PMC3989344?pdf=render

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.