Toward a mechanism-based fluorescent assay for site-specific recombinases and topoisomerases: Assay design and syntheses of fluorescent substrates

被引:5
作者
Panigrahi, G [1 ]
Zhao, BP [1 ]
Krepinsky, JJ [1 ]
Sadowski, PD [1 ]
机构
[1] UNIV TORONTO,DEPT MOL & MED GENET,TORONTO,ON M5S 1A8,CANADA
关键词
D O I
10.1021/ja9612920
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Site-specific recombinases and topoisomerases break and rejoin the phosphodiester bonds of DNA. Both classes of enzymes do so through the formation of a covalent intermediate involving a phosphodiester bond with a hydroxylated amino acid (usually tyrosine). We have previously shown that oligonucleotides that bear a 3'-phosphoryltyrosine residue linked to the phosphoryl group via a phenolic hydroxyl group are effective substrates for the assay of ligation by the FLP recombinase and mammalian topoisomerase I. In this article we describe the synthesis of oligonucleotides bearing several novel 3'-phosphoryl substituents. It is shown that oligonucleotides bearing a 3'-phosphoryltyrosine residue N-substituted on tyrosine with the bulky fluorescent groups dansyl and pyrene are ligated effectively by the FLP recombinase, and the dansyltyrosine derivative is used as effectively as the tyrosine adduct by mammalian topoisomerase I. The dansyl derivatives were completely stable during the syntheses; this underlines the potential usefulness of the dansylated class of compounds for the development of simplified assays and for mechanistic studies of breaking-joining enzymes.
引用
收藏
页码:12004 / 12011
页数:8
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