Abstract:

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.