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The effect of sequence context on the activity of cytosine DNA glycosylases

The effect of sequence context on the activity of cytosine DNA glycosylases
The effect of sequence context on the activity of cytosine DNA glycosylases
We have prepared single (N204D) and double (N204D:L272A) mutants of human uracil DNA glycosylase (hUDG), generating two cytosine DNA glycosylases (hCDG and hCYDG). Both these enzymes are able to excise cytosine (but not 5-methylcytosine), when this base is part of a mismatched base pair. hCDG is more active than the equivalent E. coli enzyme (eCYDG) and also has some activity when the cytosine is paired with guanine, unlike eCYDG. hCDG also has some activity against single stranded DNA, while having poor activity towards an unnatural base pair that forces the cytosine into an extrahelical conformation (in contrast to eCYDG for which a bulky base enhances the enzyme's activity). We also examined how sequence context affects the activity of these enzymes, determining the effect of flanking base pairs on cleavage efficiency. An abasic site or a hexaethylene glycol linker placed opposite the target cytosine, also causes an increase in activity compared with an AC mismatch. Flanking an AC mismatch with GC base pairs resulted in a 100-fold decrease in excision activity relative to flanking AT base pairs and the 5′-flanking base pair had a greater effect on the rate of cleavage. However, this effect is not simply due to the stability of the flanking base pairs as adjacent GT mismatches also produce low cleavage efficiency.
1742-206X
3273-3278
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. (2015) The effect of sequence context on the activity of cytosine DNA glycosylases. Molecular BioSystems, 11 (12), 3273-3278. (doi:10.1039/C5MB00532A).

Record type: Article

Abstract

We have prepared single (N204D) and double (N204D:L272A) mutants of human uracil DNA glycosylase (hUDG), generating two cytosine DNA glycosylases (hCDG and hCYDG). Both these enzymes are able to excise cytosine (but not 5-methylcytosine), when this base is part of a mismatched base pair. hCDG is more active than the equivalent E. coli enzyme (eCYDG) and also has some activity when the cytosine is paired with guanine, unlike eCYDG. hCDG also has some activity against single stranded DNA, while having poor activity towards an unnatural base pair that forces the cytosine into an extrahelical conformation (in contrast to eCYDG for which a bulky base enhances the enzyme's activity). We also examined how sequence context affects the activity of these enzymes, determining the effect of flanking base pairs on cleavage efficiency. An abasic site or a hexaethylene glycol linker placed opposite the target cytosine, also causes an increase in activity compared with an AC mismatch. Flanking an AC mismatch with GC base pairs resulted in a 100-fold decrease in excision activity relative to flanking AT base pairs and the 5′-flanking base pair had a greater effect on the rate of cleavage. However, this effect is not simply due to the stability of the flanking base pairs as adjacent GT mismatches also produce low cleavage efficiency.

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More information

Accepted/In Press date: 4 October 2015
e-pub ahead of print date: 7 October 2015
Published date: 1 December 2015
Organisations: Molecular and Cellular, Chemistry, Molecular Diagnostics & Thereputics

Identifiers

Local EPrints ID: 408769
URI: http://eprints.soton.ac.uk/id/eprint/408769
ISSN: 1742-206X
PURE UUID: 16a79e9d-065d-4f7c-8563-8ada613e0f43
ORCID for Keith R. Fox: ORCID iD orcid.org/0000-0002-2925-7315

Catalogue record

Date deposited: 27 May 2017 04:03
Last modified: 20 Jul 2019 01:27

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