Generation of Fluorescent Versions of Saccharomyces cerevisiae RPA to Study the Conformational Dynamics of Its ssDNA-Binding Domains.
Generation of Fluorescent Versions of Saccharomyces cerevisiae RPA to Study the Conformational Dynamics of Its ssDNA-Binding Domains.
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DOI：
10.1007/978-1-0716-1290-3_9
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Antony E
中科院分区：
文献类型：
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作者：
Kuppa S;Pokhrel N;Corless E;Origanti S;Antony E
复制蛋白 A (RPA) 是一种重要的单链 DNA (ssDNA) 结合蛋白，可隔离 ssDNA 并保护其免遭溶核降解。 RPA-ssDNA 核蛋白充当枢纽，将二十多种 DNA 代谢酶招募到 ssDNA 上，以协调 DNA 复制、修复和重组。 RPA 作为由 RPA70、RPA32 和 RPA14 亚基组成的异源三聚体发挥作用，并具有多个 DNA 结合和蛋白质相互作用结构域。其中几个结构域通过无序连接体连接，使 RPA 能够在 ssDNA 上采用多种构象。在这里，我们描述了一种基于荧光的工具来监测 RPA 的选定 DNA 结合域的动态。利用非规范氨基酸对在 BL21 (DE3) 及其衍生物中异源表达的酿酒酵母 RPA 中的荧光探针进行位点特异性改造。还描述了合成非规范氨基酸 4-叠氮基-1-苯丙氨酸 (4AZP) 的程序。详细介绍了与 ssDNA 结合后产生可测量的荧光变化的荧光团定位位点。这种通过非规范氨基酸 (FEncAA) 方法实现的荧光增强也可应用于其他 DNA 结合蛋白，以研究蛋白质-核酸相互作用的动态。
Replication protein A (RPA) is an essential single-stranded DNA (ssDNA)-binding protein that sequesters ssDNA and protects it from nucleolytic degradation. The RPA-ssDNA nucleoprotein acts as a hub to recruit over two dozen DNA metabolic enzymes onto ssDNA to coordinate DNA replication, repair, and recombination. RPA functions as a heterotrimer composed of RPA70, RPA32, and RPA14 subunits and has multiple DNA-binding and protein-interaction domains. Several of these domains are connected by disordered linkers allowing RPA to adopt a wide variety of conformations on ssDNA. Here we describe a fluorescence-based tool to monitor the dynamics of select DNA-binding domains of RPA. Noncanonical amino acids are utilized to site-specifically engineer fluorescent probes in Saccharomyces cerevisiae RPA heterologously expressed in BL21 (DE3) and its derivatives. A procedure to synthesize 4-azido-l-phenylalanine (4AZP), a noncanonical amino acid, is also described. Sites for fluorophore positioning that produce a measurable change in fluorescence upon binding to ssDNA are detailed. This fluorescence enhancement through noncanonical amino acid (FEncAA) approach can also be applied to other DNA-binding proteins to investigate the dynamics of protein-nucleic acid interactions.