Biochemistry Of DNA Repair And Transcription

DNA修复和转录的生物化学

• 批准号: 7132321
• 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/7132321
• 关键词: DNA repair; T cell receptor; adenine; cell line; cytogenetics; developmental genetics; dwarfism; enzyme activity; enzyme substrate; genetic disorder; genetic transcription; guanine; helicase; molecular genetics; nonvisual photosensitivity; oxidative stress; pentosyltransferase; premature aging; protein protein interaction

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 (TCR). 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 phenotypes of CS include a significantly increased sensitivity to a number of DNA-damaging agents including UV irradiation. 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 base excision repair (BER) there is an incision in DNA 5' to the lesion. The incision can be quantified in cell extracts by using oligonucleotide duplexes that contain a single 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 and its 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. The role of CSB in the base excision repair pathway is further supported by our recent results concerning CSB protein interactions. In searching for proteins that bind and functionally interact with CSB we have shown that a major protein binder is PARP-1, a protein involved in the initial phases of DNA single strand break repair. CSB is a substrate for PARP-1 ribosylation and it is likely that these two proteins function together in the process of base excision.

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。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（TCR）的优先修复和链特异性修复中有缺陷。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许多DNA损伤剂（包括UV照射）的显著增加的敏感性。将CSB基因转染到具有CS-B表型的仓鼠细胞中，可以将TCR和紫外线抗性完全恢复到正常水平，这表明CS-B中TCR的缺陷是由于该基因的突变造成的。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。我们已经使用体外测定来测量DNA修复过程的切口事件。在碱基切除修复（BER）的第一步中，在损伤的DNA 5'中存在切口。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损伤及其修复中的直接作用。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。CSB在碱基切除修复途径中的作用得到了我们最近关于CSB蛋白相互作用的结果的进一步支持。在寻找与CSB结合并在功能上相互作用的蛋白质时，我们已经表明主要的蛋白质结合剂是PARP-1，一种参与DNA单链断裂修复初始阶段的蛋白质。CSB是PARP-1核糖基化的底物，并且这两种蛋白质可能在碱基切除过程中一起起作用。

项目成果

Primary fibroblasts of Cockayne syndrome patients are defective in cellular repair of 8-hydroxyguanine and 8-hydroxyadenine resulting from oxidative stress.

Cockayne 综合征患者的原代成纤维细胞因氧化应激而导致 8-羟基鸟嘌呤和 8-羟基腺嘌呤的细胞修复存在缺陷。

• DOI: 10.1096/fj.02-0851com
• 发表时间: 2003
• 期刊: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: Tuo,Jingsheng;Jaruga,Pawel;Rodriguez,Henry;Bohr,VilhelmA;Dizdaroglu,Miral
• 通讯作者: Dizdaroglu,Miral

Toxicological Profiling and Long-Term Effects of Bare, PEGylated- and Galacto-Oligosaccharide-Functionalized Mesoporous Silica Nanoparticles.

• DOI: 10.3390/ijms242216158
• 发表时间: 2023-11-10
• 期刊: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
• 影响因子: 5.6
• 作者: Barguilla, Irene;Candela-Noguera, Vicente;Oliver, Patrick;Annangi, Balasubramanyam;Diez, Paula;Aznar, Elena;Martinez-Manez, Ramon;Marcos, Ricard;Hernandez, Alba;Marcos, Maria Dolores
• 通讯作者: Marcos, Maria Dolores