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

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

1932-6203
e95394
Kimber, Scott T.
912df707-c67a-42eb-8f6d-5395e7397d38
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Fox, Keith R.
9da5debc-4e45-473e-ab8c-550d1104659f
Kimber, Scott T.
912df707-c67a-42eb-8f6d-5395e7397d38
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Fox, Keith R.
9da5debc-4e45-473e-ab8c-550d1104659f

Kimber, Scott T., Brown, Tom and Fox, Keith R. (2014) A mutant of uracil DNA glycosylase that distinguishes between cytosine and 5-methylcytosine. PLoS ONE, 9 (4), e95394. (doi:10.1371/journal.pone.0095394).

Record type: Article

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.

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Accepted/In Press date: 25 March 2014
Published date: 16 April 2014
Organisations: Molecular and Cellular

Identifiers

Local EPrints ID: 364303
URI: https://eprints.soton.ac.uk/id/eprint/364303
ISSN: 1932-6203
PURE UUID: f80ec6ec-6907-4991-9f85-ad60ee53c5c9
ORCID for Keith R. Fox: ORCID iD orcid.org/0000-0002-2925-7315

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Date deposited: 22 Apr 2014 10:09
Last modified: 22 Jan 2019 01:39

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