The p53 tumor suppressor gene is a critical component of the mammalian cellular response to DNA damage. Mutations or inactivation of wild-type p53 are common in human cancers. Wild-type p53 is involved in the regulation of cell cycle checkpoints and apoptosis following DNA damage, and loss of p53 activity promotes mutagenesis and genetic instability. Recently, an additional cellular role for p53 has been described, for the regulation of DNA repair following UV-irradiation. Cells mutant for p53 exhibit defect global nucleotide excision repair of UV-induced DNA photoproducts from genomic DNA, but maintain the preferential transcription-coupled DNA repair of the transcribed strand of expressed genes. The goal of the project is to determine the mechanism for p53-dependent DNA repair. The hypothesis is that p53 induces global genomic nucleotide excision repair following DNA damage through transcriptional regulation of downstream effector.
The specific aims that will be addressed are the following: 1. To determine if the products of genes known to be transcriptionally regulated by p53 are involved in DNA repair. Human and mouse genetic models with altered expression or activity of p53 regulated genes will be examined for global genomic and transcription-coupled nucleotide excision repair. 2. To determine if p53 regulates the expression of genes whose products are known to be involved in nucleotide excision repair, and through what molecular mechanism. Quantitative analysis of UV-inducible and p53- regulated DNA repair gene expression will be performed using RT-PCR and immunoblotting. Promoter-reporter assays will be used to probe the mechanism of gene regulation. 3. To identify or discover novel DNA damage-inducible and p53- regulated gene products using cDNA microarray genomics analysis. Additional genes potentially involved in p53-regulated DNA repair will be identified using whole genome expression analysis and genetically defined cell lines. 4. To determine if candidate genes involved in p53-dependent DNA repair functionally complement nucleotide excision repair when expressed in p53 null cells. Genes identified as candidates for p53- dependent DNA repair will be transfected and expressed in p53 null cells, and DNA repair activity determined following UV-irradiation.
