Mutagenesis of critical genes underlies the development of cancer and of genetic diseases. One of the major guardians against mutagenesis is the nucleotide excision repair (NER) system that repairs single base DNA adducts in an error free manner. Little is known about the activity of NER on more extensive alterations in DNA structure. The investigator has recently reported data that suggests that the NER system can recognize and process normal DNA bases present in the distorted, alternate conformation of a triple helix, a structure that is known to block transcription. In contrast to repair of conventional adducts, the process is error prone. The triplex induced mutagenesis required the function of transcription coupled repair, TCR, which is involved in the preferential repair of transcribed DNA. These data are consistent with the proposal that sequences that block transcription could trigger """"""""gratuitous repair induced mutagenesis."""""""" Dr. Seidman proposes to use the triple helix as a novel structural probe of the NER system. The applicant will exploit the flexibility of the supF shuttle vector system and construct custom marker genes which will facilitate examination of the influence of triplex structural features on recognition by the NER system. The requirement for transcription through the triplex region for NER recognition will be determined. Then human cell lines with specific defects in repair genes will be used to define the role of individual components of the NER system in recognition and processing of triplex distortion.
