Unusual DNA-DNA cross-links between a photolyase deoxyribozyme, UV1C, and its bound oligonucleotide substrate.
UV1C is a photolyase deoxyribozyme that repairs thymine dimers in a DNA oligonucleotide substrate. We report that treatment with iodine generates specific DNA-DNA cross-links between UV1C and a bound substrate analogue, LDPs, in which a single phosphate at the photoreactivation site has been replaced with a phosphorothioate. Although iodine has been reported to generate lysine-cysteine cross-links within a protein, the formation of DNA-DNA cross-links is both unexpected and novel. We have used different mapping procedures to identify a number of bases located in loops of the G-quadruplex fold of UV1C as the sites for cross-linking with LDPs. Mutation of one cross-linking adenine, in particular, leads to a major reduction in UVIC's catalytic activity. A map of these contact cross-linking sites enables us to refine an earlier structural-topological model for the folded UV1C.LDPs complex. The surprising facility with which these novel contact cross-links can be generated between a nucleic acid enzyme and its substrate's reaction site opens up a powerful new approach to mapping the active sites of different ribozymes and deoxyribozymes as well as enabling the dissection of key contacts within RNA-protein complexes.
Sekhon
Gurpreet S
GS
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
Sen
Dipankar
D
D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000737:chemistry*; D021881:DNA, Catalytic / Q000737:chemistry*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013379:Substrate Specificity
D021881:DNA, Catalytic; D009838:Oligodeoxyribonucleotides; D004247:DNA; D004255:Deoxyribodipyrimidine Photo-Lyase