BIOCHEMISTRY OF DNA REPAIR AND TRANSCRIPTION

DNA 修复和转录的生物化学

• 批准号: 6160486
• 负责人: V A BOHR
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6160486
• 关键词: DNA directed RNA polymerase; DNA repair; RNA biosynthesis; biochemistry; cell free system; enzyme activity; genetic transcription; phosphorylation; protein kinase; ultraviolet radiation

Summary of work: Cockayne syndrome (CS) is a rare human disease that is characterized by arrested post-natal growth and resulted in premature aging and death. Cells from CS individuals are abnormally sensitive to killing by ultraviolet radiation as well as certain so-called UV-mimetic chemicals, such as 4-nitroquinoline-1-oxide and N-acetoxy-2-acetylaminofluorene. This cellular phenotype prompted extensive studies on the ability of CS cells to carry out nucleotide excision repair both in intact cells and in cell-free systems. Most conventional assays, including the use a cell-free system that supports transcription-independent nucleotide excision repair, indicate no defect in CS cells. However, CS cells are defective in the enhanced rate of repair of the template (transcribed) strand relative to the coding (non-transcribed) strand of transcriptionally active genes. In recent experiments from this laboratory we have demonstrated that mutations in the CSB gene are the cause of the transcription coupled repair defect. In hamster cells homologous to CSB we can transfect with a normal CSB gene and complement the repair defect. The mechanism of TCR in eukaryotes remains to be elucidated, and the CSB protein appears. These observations and discovery of dual function of transcription factor II H (TFIIH) in transcription and DNA repair, led to the "transcriptional hypothesis" which postulates that transcription defects are the underlying basis for the pathology in some human diseases including Cockayne syndrome. In this laboratory we were interested to test this hypothesis experimentally, and we have demonstrated the reduced level of RNA polymerase II (Pol II) transcription in intact and permeabilized CS-B cells. The molecular mechanism responsible for this deficiency was further investigated in a cell-free system. We utilized in vitro transcription assay and determine Pol II transcription activity in extracts prepared from different CS cell lines in comparison to extracts prepared from normal cells. We found that in vitro transcription is CS extracts is highly sensitive to minor damages in template DNA arising during purification. This deficiency may be complemented by transfection of a CS-B cell line with normal CSB gene. Studies of transcription in vitro in a plasmid based system demonstrate a significant transcription defect in CSB cells. This defect may be related to oxidation damage or structural changes in the DNA which somehow affects the transcription in CSB cells but not in normal cells. Experiments in intact cells also demonstrate a defect in basal transcription which can be complemented by transfection with the normal CSB gene. Further, these experiments suggest that CSB cells may have a defect in the assembly of the higher order chromatin structural organization in conjunction with transcription and DNA repair. This is supported by the observation that CSB chromatin is much more sensitive to detergent than normal chromatin.

工作总结： Cockayne综合征（CS）是一种罕见的人类疾病， 其特征是产后生长受阻， 衰老和死亡来自CS个体的细胞对以下物质异常敏感： 紫外线辐射以及某些所谓的紫外线模拟物 化学品，如4-硝基喹啉-1-氧化物和 N-乙酰氧基-2-乙酰氨基芴。这种细胞表型促使 对CS细胞进行核苷酸转移的能力的广泛研究 在完整细胞和无细胞系统中的切除修复。最 常规测定，包括使用无细胞系统， 非转录依赖性核苷酸切除修复，表明无缺陷 在CS细胞中。然而，CS细胞在增强的细胞增殖率方面是有缺陷的。 模板（转录）链相对于编码 转录活性基因的（非转录的）链。近几 我们在这个实验室的实验表明， CSB基因是转录偶联修复缺陷的原因。 在 与CSB同源的仓鼠细胞，我们可以用正常的CSB基因转染 并补充修复缺陷。真核生物TCR的作用机制 尚待阐明，CSB蛋白出现。 这些观察和转录因子双重功能的发现 II H（TFIIH）在转录和DNA修复中的作用，导致了"转录 该假说假定转录缺陷是 包括Cockayne在内的一些人类疾病的病理学基础 综合征 在这个实验室里，我们有兴趣测试这个假设， 实验上，我们已经证明了RNA水平的降低， 完整和透化CS-B中聚合酶II（Pol II）转录 细胞对这种缺陷的分子机制进行了进一步的研究。 在无细胞系统中研究。我们利用体外转录 分析和确定制备的提取物中Pol II转录活性 与不同CS细胞系制备的浸提液相比， 正常细胞我们发现，在体外转录是CS提取物， 对模板DNA中的微小损伤高度敏感， 洁净.这种缺陷可以通过转染一种 具有正常CSB基因的CS-B细胞系。 体外转录研究 基于质粒的系统显示出在以下方面的显著转录缺陷： CSB细胞。 这种缺陷可能与氧化损伤或结构损伤有关。 在某种程度上影响CSB细胞转录的DNA变化 而不是在正常细胞中。 在完整细胞中的实验也证明了 可通过转染补充的基础转录缺陷 有正常的CSB基因 此外，这些实验表明， 细胞可能在高级染色质的组装中有缺陷 结构组织与转录和DNA修复结合。 这一点得到了以下观察结果的支持，即CSB染色质比CSB染色质多得多。 比正常染色质对去污剂更敏感。