Preparation of the Modular Multi-Domain Protein RPA for Study by NMR Spectroscopy
Preparation of the Modular Multi-Domain Protein RPA for Study by NMR Spectroscopy
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DOI：
10.1007/978-1-61779-480-3_11
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发表时间：
2012-01-01
期刊：
影响因子：
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通讯作者：
Chazin, Walter J.
中科院分区：
文献类型：
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作者：
Brosey, Chris A.;Chagot, Marie-Eve;Chazin, Walter J.
基因组的完整性和繁殖取决于DNA加工事件的保真度，如复制，损伤识别和修复。DNA加工所需的许多生物化学任务的必要条件是单链DNA（ssDNA）的产生和操作。复制蛋白A（RPA）是真核生物中主要的单链DNA结合蛋白，它能保护单链DNA模板不受杂散核酸酶的切割和过早的退火。更重要的是，RPA还可以作为一个平台，用于组织访问ssDNA，以读取遗传密码，识别DNA中的畸变，并通过酶进行处理。我们已经提出，RPA的能力，以适应这样一个广泛的多蛋白质机器部分来自其模块化的组织和域间的灵活性。虽然是功能所必需的，但RPA的模块化灵活性对提供全长蛋白质的动态结构的详细表征提出了许多挑战。为了使RPA的域间动力学和响应ssDNA结合的生物物理方法，包括NMR光谱学的研究，我们已经成功地产生了重组全长RPA毫克量的天然丰度和丰富的NMR活性同位素。
The integrity and propagation of the genome depend upon the fidelity of DNA processing events, such as replication, damage recognition, and repair. Requisite to the numerous biochemical tasks required for DNA processing is the generation and manipulation of single-stranded DNA (ssDNA). As the primary eukaryotic ssDNA-binding protein, Replication Protein A (RPA) protects ssDNA templates from stray nuclease cleavage and untimely reannealment. More importantly, RPA also serves as a platform for organizing access to ssDNA for readout of the genetic code, recognition of aberrations in DNA, and processing by enzymes. We have proposed that RPA's ability to adapt to such a broad spectrum of multiprotein machinery arises in part from its modular organization and interdomain flexibility. While requisite for function, RPA's modular flexibility has presented many challenges to providing a detailed characterization of the dynamic architecture of the full-length protein. To enable the study of RPA's interdomain dynamics and responses to ssDNA binding by biophysical methods including NMR spectroscopy, we have successfully produced recombinant full-length RPA in milligram quantities at natural abundance and enriched with NMR-active isotopes.