Base Excision DNA Repair in Premature Aging and Neurodegeneration

过早衰老和神经退行性疾病中的碱基切除 DNA 修复

• 批准号: 8148312
• 负责人: David Wilson
• 金额: $ 26.94万
• 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/8148312
• 关键词: Base Excision Repairs; Nerve Degeneration; Premature aging syndrome

Cockayne Syndrome (CS) is an autosomal recessive disorder, characterized by growth failure, neurological abnormalities, premature aging symptoms, and cutaneous photosensitivity, but no increased cancer incidence. CS is divided into two complementation groups: CSA (mutation in CKN1) and CSB (mutation in ERCC6). Of the patients suffering from CS, 80% have mutations in the CSB gene. We are pursuing the hypothesis that the primary role of CS proteins is to facilitate the repair of endogenous DNA damage, and we have evidence for a direct role of CSB in regulating BER efficiency. In addition, our recent work has revealed that Ca2+ is a novel metal cofactor for CSB catalyzed DNA-dependent ATP hydrolysis, but that CSB lacks detectable ATP dependent helicase and single- or double-stranded nucleic acid translocase activities in the presence of either Ca2+ or Mg2+. We have also discovered that (i) CSB supports ATP independent complementary strand annealing of not only DNA/DNA duplexes, but DNA/RNA and RNA/RNA duplexes; (ii) CSB forms a stable protein:DNA complex with a 34mer double-stranded DNA in electrophoretic mobility shift assays, independent of divalent metal or nucleotide (e.g. ATP); and (iii) CSB stably binds a range of nucleic acid substrates, including bubble and pseudo-triplex double-stranded DNAs that resemble replication and transcription intermediates, as well as forked duplexes of DNA/DNA, DNA/RNA, and RNA/RNA composition, the latter two of which do not promote CSB ATPase activity. Moreover, association of CSB with DNA, independent of ATP binding or hydrolysis, was found to displace or rearrange a stable pre-bound protein:DNA complex, a property likely important for its roles in transcription and DNA repair. More recent results, obtained in collaboration with Dr. Vilhelm Bohr, suggest that CSB plays a direct role in not only nuclear BER, but in mitochondrial BER, likely by helping recruit, stabilize, and/or retain BER proteins in repair complexes associated with the inner mitochondrial membrane. Future work will continue to examine the in vitro activities of CSB on key DNA and RNA transaction intermediates, and will elucidate the contributions of the unique N- and C-terminal portions of the protein that likely impart functional specificity. Huntington disease (HD) is a neurodegenerative disorder that belongs to a large family of genetic diseases caused by abnormal expansion of CAG/CTG repetitive sequences. Trinucleotide repeat expansions are unstable in the genome, both in germline and somatic cells. Expansion events in both cell types have deleterious clinical consequences in HD. For instance, transmission of longer repeats to offspring results in an earlier onset of disease, where extensive somatic expansion in the striatum, the brain region primarily affected in HD, is proposed to accelerate disease pathology. Thus, understanding the mechanisms of trinucleotide repeat instability is a major interest. We have recently found that oxidative DNA lesions abnormally accumulate at CAG expansions in a length-dependent, yet age- and tissue-independent, manner, likely due to the secondary structures formed by CAG repeats that limit access of enzymes that initiate BER. In addition, our data indicate that repair by BER enzymes of some of the accessible lesions results in somatic expansion when the ratio of FEN1 to POL is low, as found to occur in the striatum. Our results therefore support BER enzyme stoichiometry as a contributor to the tissue selectivity of somatic CAG expansion in HD, a hypothesis that we are currently pursuing in greater detail. XRCC1 is a critical scaffold protein that orchestrates efficient single-strand break repair (SSBR). Recent data has found an association of XRCC1 with proteins causally linked to human spinocerebellar ataxias - aprataxin and tyrosyl-DNA phosphodiesterase 1 - implicating SSBR in protection against neuronal cell loss and neurodegenerative disease. We have found that (i) shRNA lentiviral-mediated XRCC1 knockdown in human SH-SY5Y neuroblastoma cells results in a largely selective increase in sensitivity of the nondividing (i.e. terminally differentiated) cell population to the oxidizing agents, menadione and paraquat, and (ii) primary XRCC1 heterozygous mouse cerebellar granule cells or primary human fetal brain neurons depleted for XRCC1 exhibit increased strand break accumulation and reduced survival following menadione treatment. We are currently assessing the role of this protein in age-related pathologies using heterozygous mice, and will concomitantly evaluate the effect of XRCC1 haploinsufficiency on neurodegeneration and cancer proneness following defined insults. Finally, investigations exploring the possible role of aprataxin and tyrosyl-DNA phosphodiesterase 1 in maintaining mitochondrial function are ongoing.

Cockayne综合征（CS）是一种常染色体隐性疾病，其特征是生长衰竭，神经系统异常，过早衰老症状和皮肤光敏性，但癌症发病率没有增加。 CS分为两个互补组：CSA（CKN1中的突变）和CSB（ERCC6中的突变）。 在患有CS的患者中，有80％的患者患有CSB基因突变。 我们追求的假设是，CS蛋白的主要作用是促进内源性DNA损伤的修复，并且我们有证据表明CSB在调节BER效率方面的直接作用。 此外，我们最近的工作表明，Ca2+是CSB催化DNA依赖性ATP水解的新型金属辅因子，但是CSB缺乏在Ca2+或Mg2+的存在下，CSB缺乏可检测到的ATP依赖性解旋酶和单链或双链核酸易位酶活性。 我们还发现（i）CSB不仅支持DNA/DNA双链体的ATP独立互补链退火，还支持DNA/RNA和RNA/RNA/RNA双链体； （ii）CSB形成了一种稳定的蛋白质：DNA复合物，具有34mer双链DNA在电泳迁移率转移测定中，与二价金属或核苷酸无关（例如ATP）； （iii）CSB稳定地结合了一系列核酸底物，包括类似于复制和转录中间体的气泡和伪三链双链DNA，以及DNA/DNA的分叉复式，DNA/DNA/RNA/RNA/RNA/RNA/RNA/RNA组成，并未促进CSB的活性CSB。 此外，发现CSB与DNA的缔合，独立于ATP结合或水解，可以取代或重新排列稳定的预组结构蛋白：DNA复合物，DNA复合物，这对于其在转录和DNA修复中的作用可能很重要。 与Vilhelm Bohr博士合作获得的最新结果表明，CSB不仅在核BER中起着直接的作用，而且在线粒体BER中起着直接的作用，这可能是通过帮助募集，稳定和/或保留与内部线粒体膜相关的维修复合物中的招募，稳定和/或保留BER蛋白。 未来的工作将继续检查CSB对关键DNA和RNA交易中间体的体外活性，并将阐明蛋白质的独特N和C末端部分的贡献，这些蛋白可能会赋予功能特异性。 亨廷顿疾病（HD）是一种神经退行性疾病，属于由CAG/CTG重复序列异常扩张引起的大型遗传疾病家族。 在种系和体细胞中，基因组中的三核苷酸重复膨胀是不稳定的。 两种细胞类型的扩展事件在HD中都有有害的临床后果。 例如，将较长的重复传播到后代会导致疾病的早期发作，在这种疾病中，纹状体的大量体细胞扩张（主要在HD中受到HD影响）的大量扩张是为了加速疾病病理。 因此，了解三核苷酸重复不稳定性的机制是主要兴趣。 我们最近发现，氧化性DNA病变异常在CAG膨胀处积累，但依赖性和组织不依赖的方式，可能是由于CAG重复出现的二级结构所致，从而限制了限制启动BER的酶的访问。 此外，我们的数据表明，当Fen1与POL的比率较低时，通过BER酶修复某些可及性病变会导致体细胞膨胀，这是在纹状体中发生的。 因此，我们的结果支持BER酶化学计量学是HD中体细胞CAG扩展的组织选择性的促进者，这是我们目前正在更详细地追求的假设。 XRCC1是一种关键的脚手架蛋白，它可以编排有效的单链破裂修复（SSBR）。最近的数据发现，XRCC1与蛋白质的关系与人脊椎发子共济失调有关 - 阿普拉天蛋白和酪糖基-DNA磷酸二酯酶1-与SSBR有关保护神经元细胞丧失和神经退行性疾病。 我们已经发现，（i）shRNA慢病毒介导的人类SH-SY5Y神经母细胞瘤细胞中的XRCC1敲低导致非分散敏感性（即终端分化）对氧化剂，麦芽麦酸酯和（paraquat和paraquat和（ii）Xrcc1 xrcc1 heles ofter gran Gran eyl eyzy eyl offer fimem core n键（即终端区分）细胞种群的敏感性大大提高，并且XRCC1耗尽的神经元表现出增加链断裂的积累，而在Menadione治疗后的存活率降低。 我们目前正在使用杂合小鼠评估该蛋白在与年龄相关的病理学中的作用，并将同时评估XRCC1单倍不足性对神经变性和癌症的影响，而定义侮辱后。 最后，探索Aprataxin和酪酶-DNA磷酸二酯酶1在维持线粒体功能中的可能作用的研究正在进行中。