Trp repressor interaction with bromodeoxyuridine-substituted operators alters UV-induced perturbation pattern in a sequence-dependent manner

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In order to explore DNA sites influenced by the trp repressor-operator interaction, bromodeoxyuridine (BrdU) was chemically incorporated into the TrpEDCBA, TrpR, and aroH operators at selected thymidine positions. Different patterns of repressor protection from strand scission in the two halves of complexes with the TrpEDCBA, TrpR, and aroH operators suggest different local environments despite the highly symmetric sequences. Although protection was observed at multiple sites in the operators in the presence of repressor, UV irradiation did not lead to a cross-linked repressor-operator complex. This result indicates the absence of close contacts in the major groove between suitable repressor residues and the 5-methyl of thymidines. Upon trp repressor binding and UV irradiation, in addition to protection from strand scission, multiplets were observed at some sites, notably within CTAG sequences in the BrdU-substituted operators. This phenomenon (termed band migration) may result from distortion by the trp repressor of the BrdU-substituted operator DNA and consequent exposure of different sites along the backbone to strand scission. Interestingly, UV footprinting of two BrdU-substituted TrpEDCBA variant operators showed different patterns when base pair symmetry was matched to each side of the symmetry axis. These observations suggest that alterations in the UV photolysis pattern in response to protein binding result from DNA structural alterations that are sequence dependent.