Fluorescent human RPA to track assembly dynamics on DNA.
Fluorescent human RPA to track assembly dynamics on DNA.
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荧光人类 RPA 用于追踪 DNA 上的组装动态。
DOI：
10.1101/2023.11.23.568455
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Antony,Edwin
中科院分区：
文献类型：
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作者：
Kaushik,Vikas;Chadda,Rahul;Kuppa,Sahiti;Pokhrel,Nilisha;Vayyeti,Abhinav;Grady,Scott;Arnatt,Chris;Antony,Edwin
DNA代谢过程包括复制、修复、重组和端粒维持发生在单链DNA（ssDNA）上。在每一个复杂的过程中，几十种蛋白质在ssDNA模板上共同发挥作用。然而，当双链DNA解绕时，瞬时开放的ssDNA被高亲和力异源三聚体ssDNA结合复制蛋白A（RPA）保护和包被。几乎所有的下游DNA过程必须首先重塑/去除RPA或同时发挥作用以接近RPA下封闭的ssDNA。RPA-ssDNA复合物的形成触发DNA损伤检查点反应，并且是激活大多数DNA修复和重组途径的关键步骤。因此，除了保护暴露的ssDNA之外，RPA还充当守门人以定义DNA维持和基因组完整性中的功能特异性。RPA通过多结构域结构实现功能灵活性，该结构域利用由柔性接头连接的几个DNA结合和蛋白质相互作用结构域。这种灵活的模块化架构使RPA能够采用针对特定DNA代谢作用量身定制的各种配置。为了实验性地捕获RPA结构域与ssDNA和相互作用蛋白结合后的动态变化，我们在这里描述了使用4-叠氮基-L-苯丙氨酸（4AZP）掺入和点击化学产生活性位点特异性荧光形式的人RPA（RPA）。这种方法也可以应用于其他多结构域蛋白的位点特异性修饰。还描述了通过非典型氨基酸（FEncAA）和Förster共振能量转移（FRET）测定RPA对DNA的动力学的增强作用。本文描述的荧光人RPA将使RPA-ssDNA相互作用的高分辨率结构-功能分析成为可能。
DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. The fluorescent human RPA described here will enable high-resolution structure-function analysis of RPA-ssDNA interactions.