In situ detection of stalled cleavage complexes for studies in aging

用于老化研究的停滞裂解复合物的原位检测 摘要 本项目将介绍和验证第一个定量原位方法检测停滞的共价切割 复合物（SCC），其在哺乳动物细胞的正常衰老期间自发地起源于哺乳动物细胞。这些 共价拓扑异构酶-DNA链断裂中间体不断产生并缓慢积累， 老化细胞它们的形成被与年龄相关的退行性疾病加速，如阿尔茨海默氏症。 SCC是在失败的催化过程中产生的，最近被发现是老化过程的关键部分 在真核生物中。尽管SCC在正常衰老过程和病理学中起着重要作用， 与衰老相关，目前，还没有特异性的方法检测和定量这些DNA 改变在原位与个人复杂的敏感性。现有的散装生物化学方法不是 适用于原位形式，例如固定的细胞和组织切片。 在这个项目中，我们将通过开发一种新的分子生物学技术来克服这一限制。 老化分析。创新的原位方法将基于新的标记原理，并将 同时共同检测SCC几个特性特征。 这项研究将缩小一个重大的技术差距，并将引入第一个定量分析， 现场SCC评估。该项目将为衰老和年龄相关的分子研究创造一个新的工具。 疾病，在整个生物医学中具有广泛和普遍的效用。 该项目将实现这些具体目标： 1.介绍第一种定量原位检测共价停滞裂解复合物（SCC）的方法， 其在哺乳动物细胞的正常衰老过程中自发地起源于哺乳动物细胞。测试和确认标签 通过使用具有受控SCC生产的原位模型，了解新技术的原理。确保 新方法的定量能力及其对SCC的高度特异性。 2.在细胞和组织切片模型中进行生物化学和体内实验， SCC的产生。评估和确认新方法的定量能力及其高度特异性 对于SCC。确保对各种样本进行稳健可靠的检测性能。

In situ detection of stalled cleavage complexes for studies in aging Abstract This project will introduce and validate the first quantitative in situ approach detecting stalled covalent cleavage complexes (SCCs), which spontaneously originate in mammalian cells during their normal aging. These covalent topoisomerase-DNA strand break intermediates are continually produced and slowly accumulate in aging cells. Their formation is accelerated by age-related degenerative diseases, such as Alzheimer’s. SCCs are generated in abortive catalysis and were recently revealed to be a critical part of the aging process in eukaryotes. In spite of the essential role of SCCs in the normal aging process and in the pathologies associated with aging, at present time, there are no specific approaches detecting and quantitating these DNA alterations in situ with the individual complex sensitivity. The existing bulk biochemical approaches are not applicable to the in situ formats, such as fixed cells and tissue sections. In this project, we will overcome this limitation by developing a new and enabling technology for molecular analysis of aging. The innovative in situ method will be based on novel labeling principles, and will simultaneously co-detect several characteristic features of SCCs. This study will close a significant technological gap and will introduce the first assay for quantitative assessment of SCCs in situ. The project will create a new tool for molecular research in aging and age-related disorders, with broad and general utility in biomedicine at large. The project will reach these Specific Aims: 1. To introduce the first quantitative in situ approach detecting covalent stalled cleavage complexes (SCCs), which spontaneously originate in mammalian cells during their normal aging. To test and validate the labeling principles of the new technology by using in situ models with regulated production of SCCs. To ensure the quantitative ability of the new approach and its high specificity for SCCs. 2. To test and validate the new method in the cell and tissue section models with biochemical and in vivo generation of SCCs. To assess and confirm the quantitative ability of the new approach and its high specificity for SCCs. To ensure robust and reliable assay performance in various samples.