Dynamic accumulation of cyclobutane pyrimidine dimers and its response to changes in DNA conformation.

Photochemical dimerization of adjacent pyrimidines is fundamental to the creation of mutagenic hotspots caused by ultraviolet light. Distribution of the resulting lesions (cyclobutane pyrimidine dimers, CPDs) is already known to be highly variable in cells, and in vitro models have implicated DNA conformation as a major basis for this observation. Past efforts have primarily focused on mechanisms that influence CPD formation and have rarely considered contributions of CPD reversion. However, reversion is competitive under the standard conditions of 254 nm irradiation as illustrated in this report based on the dynamic response of CPDs to changes in DNA conformation. A periodic profile of CPDs was recreated in DNA held in a bent conformation by λ repressor. After linearization of this DNA, the CPD profile relaxed to its characteristic uniform distribution over a similar time of irradiation to that required to generate the initial profile. Similarly, when a T tract was released from a bent conformation, its CPD profile converted under further irradiation to that consistent with a linear T tract. This interconversion of CPDs indicates that both its formation and reversion exert control on CPD populations long before photo-steady-state conditions are achieved and suggests that the dominant sites of CPDs will evolve as DNA conformation changes in response to natural cellular processes.

Naphthalimide-Based Template for Inhibitor Screening via Cross-Linking and In-Gel Fluorescence: A Case Study against HCA II.

We describe a rapid electrophoresis-based method for profiling of carbonic anhydrase inhibitors. In addition to the pharmacophore moiety intended for reversible interaction with a target enzyme, a fluorescent template with a built-in azide group for photoaffinity labeling is also included as a part of the inhibitor design. Following incubation and irradiation, gel electrophoresis with visualization under UV allows assessment of the efficiency of cross-linking. The relative efficiency of cross-linking of various probes can be regarded as a reflection of their inhibition potencies, an assumption supported by the trend in their IC50/K i values. The method has the advantage of being applicable to impure enzyme preparations and also can be used to screen several inhibitors including their promiscuity in parallel in a short time as has been currently demonstrated with HCA II.