A contact photo-cross-linking investigation of the active site of the 8-17 deoxyribozyme.
The small RNA-cleaving 8-17 deoxyribozyme (DNAzyme) has been the subject of extensive mechanistic and structural investigation, including a number of recent single-molecule studies of its global folding. Little detailed insight exists, however, into this DNAzyme's active site; for instance, the identity of specific nucleotides that are proximal to or in contact with the scissile site in the substrate. Here, we report a systematic replacement of a number of bases within the magnesium-folded DNAzyme-substrate complex with thio- and halogen-substituted base analogues, which were then photochemically activated to generate contact cross-links within the complex. Mapping of the cross-links revealed a striking pattern of DNAzyme-substrate cross-links but an absence of significant intra-DNAzyme cross-links. Notably, the two nucleotides directly flanking the scissile phosphodiester cross-linked strongly with functionally important elements within the DNAzyme, the thymine of a G.T wobble base pair, a WCGR bulge loop, and a terminal AGC loop. Mutation of the wobble base pair to a G-C pair led to a significant folding instability of the DNAzyme-substrate complex. The cross-linking patterns obtained were used to generate a model for the DNAzyme's active site that had the substrate's scissile phosphodiester sandwiched between the DNAzyme's wobble thymine and its AGC and WCGR loops.
Liu
Yong
Y
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
Sen
Dipankar
D
D001482:Base Composition / Q000187:drug effects / Q000528:radiation effects; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects / Q000528:radiation effects; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology* / Q000528:radiation effects*; D021881:DNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D008027:Light; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation / Q000187:drug effects / Q000528:radiation effects; D010777:Photochemistry; D013379:Substrate Specificity / Q000187:drug effects / Q000528:radiation effects; D013866:Thioguanine / Q000378:metabolism
D003432:Cross-Linking Reagents; D021881:DNA, Catalytic; D013866:Thioguanine