Gene Specific DNA Repair

Bohr, Vilhelm

Abstract

of work: The nuclear matrix is a central structure in the cellular hierarchy. A number of processes have been shown to be located here, including transcription, replication and topological binding sites. There has also been documentation that DNA repair processes are preferentially located at this site. We have demonstrated that the early DNA repair processes of nucleotide excision and transcription coupled DNA repair take place here at the nuclear matrix. In response to UV irradiation of mammalian cells, the protein proliferating cell nuclear antigen (PCNA) forms an insoluble complex with nuclear substructures. This complex can be detected by immunofluorescence and western blot analysis within 30 min after UV irradiation. We have studied the role of nucleotide excision repair (NER) and its subcomponent, transcription coupled repair (TCR), in PCNA complex formation. PCNA complex formation was studied in genetically related hamster cell lines that differ only in their capacity to perform NER. We find that efficient PCNA complex formation induced by UV irradiation is dependent on both the genome overall repair of 6-4 photoproducts and the TCR of pyrimidine dimers in hamster cells. Cells from individuals with the premature aging syndrome Cockayne syndrome (CS) are also deficient in the PCNA relocation, supporting other findings from this lab demonstrating a defect of base excision repair of oxidative DNA damage in CS cells. We have biochemically analyzed the role of PCNA in the DNA repair of oxidative DNA modifications. By biochemical fractionation of cell extracts we can directly show that PCNA participates in conjunction with replication protein A (RPA) in the repair process. We have also investigated the DNA repair of photo lesions in the ribosomal DNA in relation to the RNA polymerase I transcription in the nucleoli using a combination of immunological and biochemical approaches. We find that the nucleoli are completely deficient in DNA repair despite the presence of abundant RNA pol I transcription foci. Gene specific repair assays showed that both cyclobutane pyrimidine dimers and 6-4 photoproducts are removed much less efficiently from the rDNA than from an endogenous, essential gene. This observation is further substantiated by the analysis of repair kinetics of UV induced photolesions in the isolated BudR containing repair patches isolated immunologically using antibody to BudR. Immunological staining indicates that various NER proteins are absent in nucleoli, and this may be responsible for the inefficient repair of photoproducts in the rDNA. - DNA repair, Transcription coupled repair, nuclear matrix, DNA photoproducts