Biochemistry Of DNA Repair And Transcription

DNA修复和转录的生物化学

• 批准号: 6815315
• 负责人: Vilhelm 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/6815315
• 关键词: DNA binding protein; DNA repair; RNA biosynthesis; aging; deafness; developmental genetics; dwarfism; genetic transcription; radiation genetics; retina degeneration; syndrome; tissue /cell culture; ultraviolet radiation

Summary of work: Cockayne syndrome (CS) belongs to the category of premature aging diseases, where the individuals appear much older than their chronological age. Cells from CS patients are sensitive to UV light, exhibit a delay in recovery of DNA and RNA synthesis following irradiation, and are defective in preferential repair and strand-specific repair of active genes. Complementation studies demonstrate at least two genes involved in CS, designated CSA and CSB. CSB protein, by sequence comparison, belongs to the SNF2 family of proteins, which have roles in transcriptional regulation, chromosome stability and DNA repair. The cellular and molecular phenotype of CS include a significantly increased sensitivity to a number of DNA-damaging agents including UV irradiation. Studies in CS cells were initially confined to DNA repair in the general, overall genome, where no defect was found. However, CS cells are defective in the preferential repair of active genes and in the preferential repair of the transcribed strand of such genes. Transfection of the CSB gene into hamster cells with the CS-B phenotype completely restores TCR and UV resistance to normal levels, demonstrating that the defect in TCR in CS-B is due to mutation in that gene. The complex clinical phenotype of CS, however, suggests that DNA repair may not be the only defect. We have reported a defect in basal transcription in CS both in vivo and in vitro. This transcription defect is seen in CS-B lymphoblastoid cells and fibroblasts without any exposure to stress such as UV light. We have used an in vitro assay to measure the incision event of the DNA repair process. During the first step of BER, there is an incision in DNA 5' to the lesion. The incision can be quantitated in cell extracts by using oligonucleotide duplexes that contain a single 8-oxoG and 8-oxoG lesion at a defined site. In CS-B deficient cell lines we observe a decrease in 8-oxoG incision that can be complemented by transfection of a plasmid containing the intact CSB gene. This suggests a direct role for CSB in the recognition of oxidative DNA damage. The CSB protein apparently functions at the crossroads of DNA repair and transcription. We have also observed that CS-B cells appear to have a more open chromatin structure than normal cells, what would be compatible with a role in chromatin structural assembly. It would appear that the CSB protein has more than one function and is probably involved in a large number of protein-protein interactions in transcription and repair pathways. One or more of these is likely to be very important for the assembly of the DNA repair and transcription complexes at the nuclear matrix. A functional analysis of the CSB gene has been undertaken in our laboratory to better understand the nature of the molecular deficiencies observed in CS. Mutants, generated by site- directed mutagenesis, have been tested for genetic complementation of CSB null cell lines by cell viability and RNA synthesis recovery upon exposure to UV light and other genotoxic agents. Point mutations in ATPase motifs I and II of CSB dramatically reduce CSB function in vivo, suggesting that ATP hydrolysis by CSB protein is required for transcription-coupled repair of DNA damage. This mutant also shows dramatically increased apoptosis, suggesting a role for the CS protein in the apoptotic pathway. We have now made a series of point mutations in the helicase domain of the CSB gene in stably transfected human cell lines. We find that some of these are defective in the incision of 8-oxoG, suggesting a defect in base excision repair. Further, we have analyzed the formation of another important oxidative DNA base lesion, 8-hydroxyadenine. The repair of this adduct is also deficient in CSB. We can conclude that the CSB protein is involved in the general genome base excision repair process, and that different domains of the helicase region play different roles in this process. We find that the repair defect that we observe directly correlates with cellular sensitivity to X-ray and that oxidative lesions accumulate in CSB cells after exposure. This could explain the high prevalence of neurological defects seen in CSB patients. The repair defect in CSB cells is not limited to the nuclear DNA, but also observed in mitochondrial DNA. Thus, remaining lesions in mitochondrial DNA in patients might contribute to the aging and neurodegenerative phenotypes. We have studied the function and protein interactions of the purified CSB protein. We have characterized its ATPase activity and find that it is subject to phosphorylation. The CSB protein inetracts with some of the proteins that are involved in the DNA repair of oxidative damage, the process called base excision repair. This does suggest that proficient base excision repair requires a functional CSB protein.

工作总结：Cockayne综合征（CS）属于过早衰老疾病的范畴，其中个体看起来比他们的实际年龄大得多。CS患者的细胞对紫外线敏感，照射后DNA和RNA合成恢复延迟，并且在活性基因的优先修复和链特异性修复方面存在缺陷。互补研究表明至少有两个基因参与CS，命名为CSA和CSB。通过序列比较，CSB蛋白属于SNF 2蛋白家族，其在转录调节、染色体稳定性和DNA修复中起作用。CS的细胞和分子表型包括对许多DNA损伤剂（包括UV照射）的显著增加的敏感性。最初对CS细胞的研究仅限于一般的、整个基因组的DNA修复，没有发现缺陷，然而，CS细胞在活性基因的优先修复和这些基因的转录链的优先修复方面存在缺陷。将CSB基因转染到具有CS-B表型的仓鼠细胞中完全恢复TCR和UV抗性至正常水平，表明CS-B中TCR的缺陷是由于该基因的突变。然而，CS复杂的临床表型表明DNA修复可能不是唯一的缺陷。我们已经报道了在体内和体外CS的基础转录缺陷。这种转录缺陷在CS-B淋巴母细胞样细胞和成纤维细胞中观察到，而没有任何暴露于应激如UV光。我们使用体外试验来测量DNA修复过程的切口事件。在BER的第一步中，在损伤的DNA 5'中有一个切口。可以通过使用在限定位点处含有单个8-oxoG和8-oxoG损伤的寡核苷酸双链体来定量细胞提取物中的切口。在CS-B缺陷细胞系中，我们观察到8-oxoG切口减少，其可以通过转染含有完整CSB基因的质粒来补充。这表明CSB在识别氧化DNA损伤中的直接作用。CSB蛋白显然在DNA修复和转录的十字路口起作用。我们还观察到CS-B细胞似乎比正常细胞具有更开放的染色质结构，这与染色质结构组装中的作用是相容的。CSB蛋白似乎具有不止一种功能，并且可能参与转录和修复途径中的大量蛋白质-蛋白质相互作用。其中一个或多个可能对于核基质中DNA修复和转录复合物的组装非常重要。我们实验室对CSB基因进行了功能分析，以更好地了解CS中观察到的分子缺陷的性质。通过暴露于UV光和其他遗传毒性剂后的细胞活力和RNA合成恢复，测试了通过定点诱变产生的突变体对CSB无效细胞系的遗传互补作用。CSB的ATP酶基序I和II中的点突变显著降低CSB在体内的功能，表明CSB蛋白的ATP水解是DNA损伤的转录偶联修复所必需的。该突变体还显示出显著增加的细胞凋亡，表明CS蛋白在细胞凋亡途径中的作用。我们现在已经在稳定转染的人细胞系中CSB基因的解旋酶结构域中进行了一系列点突变。我们发现，其中一些是有缺陷的8-oxoG的切口，这表明在碱基切除修复的缺陷。此外，我们分析了另一个重要的氧化DNA碱基损伤，8-羟基腺嘌呤的形成。这种加合物的修复在CSB中也是不足的。我们可以得出结论，CSB蛋白参与了一般的基因组碱基切除修复过程，并且解旋酶区域的不同结构域在此过程中发挥不同的作用。我们发现，我们观察到的修复缺陷与细胞对X射线的敏感性直接相关，并且暴露后氧化损伤在CSB细胞中积累。这可以解释CSB患者神经系统缺陷的高患病率。CSB细胞中的修复缺陷不仅限于核DNA，还在线粒体DNA中观察到。因此，患者线粒体DNA中的剩余损伤可能有助于衰老和神经退行性表型。 我们研究了纯化的CSB蛋白的功能和蛋白质相互作用。我们已经表征了它的ATP酶活性，并发现它受到磷酸化。CSB蛋白与一些参与氧化损伤DNA修复的蛋白质相互作用，该过程称为碱基切除修复。这确实表明熟练的碱基切除修复需要功能性CSB蛋白。