Panayotou G, Brown T, Barlow T, Pearl L H, Savva R
Ludwig Institute for Cancer Research, University College London, 91 Riding House Street, London, W1P 8BT, United Kingdom.
J Biol Chem. 1998 Jan 2;273(1):45-50. doi: 10.1074/jbc.273.1.45.
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.
利用表面等离子体共振技术，已使用缺乏催化活性的单纯疱疹病毒1型尿嘧啶-DNA糖基化酶的定点突变体来探究这类高度保守且普遍存在的DNA修复酶的底物识别情况。天冬氨酸-88和组氨酸-210这两个残基参与了该酶的催化机制（萨瓦，R.，麦考利-赫克特，K.，布朗，T.，以及珀尔，L.（1995年）《自然》373卷，487 - 493页；斯卢普豪格，G.，莫尔，C.D.，卡夫利，B.，阿尔瓦伊，A.S.，克罗坎，H.E.以及泰纳，J.A.（1996年）《自然》384卷，87 - 92页），分别将它们突变为天冬酰胺，以便在无催化作用的情况下研究底物识别。这些突变体被证明折叠正确且缺乏催化活性。通过使用表面等离子体共振的实时生物分子相互作用分析监测与含或不含尿嘧啶的单链和双链寡核苷酸的结合。两个突变体在相同的含尿嘧啶底物上表现出相当的结合和解离速率。发现与单链尿嘧啶-DNA的相互作用强于与双链尿嘧啶-DNA的相互作用，并且与鸟嘌呤：尿嘧啶错配的结合明显强于与腺嘌呤：尿嘧啶碱基对的结合，这表明碱基对的稳定性决定了相互作用的效率。此外，突变体与不含尿嘧啶的单链或双链DNA，或与含无碱基位点的DNA之间的相互作用可忽略不计。这些结果表明，尿嘧啶-DNA糖基化酶识别的是DNA中的尿嘧啶，而非DNA本身。
