BIROn - Birkbeck Institutional Research Online

    Direct measurement of the substrate preference of uracil-DNA glycosylase

    Panayotou, G. and Brown, T. and Barlow, T. and Pearl, L.H. and Savva, Renos (1998) Direct measurement of the substrate preference of uracil-DNA glycosylase. Journal of Biological Chemistry 273 (1), pp. 45-50. ISSN 0021-9258.

    [img]
    Preview
    Text (Refereed)
    Direct Measurement of the Substrate Preference of Uracil-DNA.pdf - Published Version of Record

    Download (137kB) | Preview

    Abstract

    Site-directed mutants of the herpes simplex virus type 1 uracil-DNA glycosylase lacking catalytic activity have been used to probe the substrate recognition of this highly conserved and ubiquitous class of DNA-repair enzyme utilizing surface plasmon resonance. The residues aspartic acid-88 and histidine-210, implicated in the catalytic mechanism of the enzyme (Savva, R., McAuley-Hecht, K., Brown, T., and Pearl, L. (1995) Nature 373, 487–493; Slupphaug, G., Mol, C. D., Kavli, B., Arvai, A. S., Krokan, H. E. and Tainer, J. A. (1996) Nature 384, 87–92) were separately mutated to asparagine to allow investigations of substrate recognition in the absence of catalysis. The mutants were shown to be correctly folded and to lack catalytic activity. Binding to single- and double-stranded oligonucleotides, with or without uracil, was monitored by real-time biomolecular interaction analysis using surface plasmon resonance. Both mutants exhibited comparable rates of binding and dissociation on the same uracil-containing substrates. Interaction with single-stranded uracil-DNA was found to be stronger than with double-stranded uracil-DNA, and the binding to Gua:Ura mismatches was significantly stronger than that to Ade:Ura base pairs suggesting that the stability of the base pair determines the efficiency of interaction. Also, there was negligible interaction between the mutants and single- or double-stranded DNA lacking uracil, or with DNA containing abasic sites. These results suggest that it is uracil in the DNA, rather than DNA itself, that is recognized by the uracil-DNA glycosylases.

    Metadata

    Item Type: Article
    School: Birkbeck Schools and Departments > School of Science > Biological Sciences
    Research Centres and Institutes: Innovation Management Research, Birkbeck Centre for
    Depositing User: Sarah Hall
    Date Deposited: 14 May 2019 11:51
    Last Modified: 02 Jul 2020 23:25
    URI: http://eprints.bbk.ac.uk/id/eprint/27527

    Statistics

    Downloads
    Activity Overview
    111Downloads
    57Hits

    Additional statistics are available via IRStats2.

    Archive Staff Only (login required)

    Edit/View Item Edit/View Item