Biochemistry Of Dna Repair And Transcription

DNA修复和转录的生物化学

• 批准号: 6530367
• 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/6530367
• 关键词: DNA repair; RNA biosynthesis; aging; deafness; developmental genetics; dwarfism; genetic transcription; premature aging; radiation genetics; retina degeneration; tissue /cell culture; ultraviolet radiation

Summary of work: Cockayne syndrome (CS) belongs to the category of premature aging disaeses 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 re-pair 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. This defect in transcription coupled repair (TCR) in CS is not only found after UV exposure but also after exposure to certain forms of oxidative stress. 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 primary 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. A previous study found that expression of a metalloprotease was reduced by 50% in CS cells, and recently it was reported that the purified CSB protein stimulates transcription, presumably as an elongation factor. 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 lesion at a defined site. In primary CS-B cell lines we observe a deficiency in incision. This deficiency can be complemented by transfection of the CS1AN (CS-B) cell line with a plasmid containing the intact CSB gene, suggesting a role for CSB in the recognition of 8-oxoG. The CSB protein apparently functions at the crossroads of DNA repair and transcription. It has been reported to interact with the structure specific incision endonuclease XPG, CSA protein, and RNA polymerase II. It has considerable homology to the SWI/SNF complex, which in yeast is associated with RNA polymerase II. The SWI/SNF complex is involved in the initiation phase of the transcription process. We have also observed that CS-B cells appear to have a more open chromatin structure than normal cells, and this would be compatible with a function that involves a role in chromatin structural assembly. It would appear that the CSB protein has more than one function and is most likely 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 factory 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. In contrast to the ATPase point mutations, deletions in the conserved acidic domain do not appear to interfere with the repair capacity of CSB protein. This suggests that this domain may be conserved in the SWI-SNF family for some other function. 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 plasmid with 8-oxoG, suggesting a defcet in base excision repair. 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.

工作总结：Cockayne综合征（CS）属于早衰性疾病的范畴，其中个体看起来比他们的实际年龄大得多。来自CS患者的细胞对UV光敏感，在照射后表现出DNA和RNA合成恢复的延迟，并且在活性基因的优先修复和链特异性重新配对方面有缺陷。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多种DNA损伤剂（包括紫外线照射）的敏感性显着增加。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。CS中转录偶联修复（TCR）的这种缺陷不仅在UV暴露后发现，而且在暴露于某些形式的氧化应激后也发现。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修复可能不是主要缺陷。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。先前的研究发现，金属蛋白酶的表达在CS细胞中减少了50%，最近有报道称，纯化的CSB蛋白刺激转录，可能是作为延伸因子。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损伤的寡核苷酸双链体在细胞提取物中定量切口。在原代CS-B细胞系中，我们观察到切口缺陷。这种缺陷可以通过用含有完整CSB基因的质粒转染CS 1AN（CS-B）细胞系来补充，表明CSB在8-oxoG的识别中的作用。CSB蛋白显然在DNA修复和转录的十字路口起作用。据报道，它与结构特异性切割核酸内切酶XPG、CSA蛋白和RNA聚合酶II相互作用。它与SWI/SNF复合物具有相当大的同源性，SWI/SNF复合物在酵母中与RNA聚合酶II相关。SWI/SNF复合物参与转录过程的起始阶段。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，这与涉及染色质结构组装的功能是相容的。CSB蛋白似乎具有不止一种功能，并且最有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DNA修复和转录工厂在核基质中的组装非常重要。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。与ATP酶点突变相反，保守酸性结构域的缺失似乎不会干扰CSB蛋白的修复能力。这表明，该结构域可能是保守的SWI-SNF家族的一些其他功能。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。我们发现其中一些在8-oxoG的质粒切割中有缺陷，表明碱基切除修复缺陷。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。