MECHANISM FOR P53-DEPENDENT DNA EXCISION REPAIR

p53肿瘤抑制基因是哺乳动物细胞对DNA损伤反应的关键组分。野生型p53的突变或失活在人类癌症中很常见。野生型p53参与DNA损伤后细胞周期检查点和细胞凋亡的调节，p53活性的丧失促进诱变和遗传不稳定性。最近，已经描述了p53的另外的细胞作用，用于调节UV照射后的DNA修复。p53突变体细胞对紫外线诱导的DNA光产物的DNA修复表现出整体核苷酸切除的缺陷，但对表达基因的转录链保持优先的转录偶联DNA修复。该项目的目标是确定p53依赖的DNA修复机制。假设p53通过下游效应子的转录调控诱导DNA损伤后的全基因组核苷酸切除修复。具体目标如下：1.确定已知受p53转录调控的基因产物是否参与DNA修复。将检查具有改变的p53调节基因的表达或活性的人类和小鼠遗传模型的全局基因组和转录偶联核苷酸切除修复。2.确定p53是否调节已知参与核苷酸切除修复的基因的表达，以及通过何种分子机制。将使用RT-PCR和免疫印迹法对UV诱导和p53调节的DNA修复基因表达进行定量分析。启动子-报告基因分析将用于探测基因调控的机制。3.利用cDNA微阵列基因组学分析鉴定或发现新的DNA损伤诱导和p53调控的基因产物。其他可能参与p53调控的DNA修复的基因将使用全基因组表达分析和遗传定义的细胞系进行鉴定。4.确定p53依赖性DNA修复中的候选基因在p53缺失细胞中表达时是否与核苷酸切除修复功能互补。将被鉴定为p53依赖性DNA修复的候选基因转染并在p53无效细胞中表达，并在UV照射后测定DNA修复活性。

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.