Section of Structural Biology, Department of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, SW7 2AZ, UK.
Department of Biological Sciences, Marquette University, Milwaukee, WI, 53201, USA.
Nat Commun. 2018 Dec 21;9(1):5447. doi: 10.1038/s41467-018-07883-7.
Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Using cryo electron microscopy we obtained a 3D reconstruction of the RPA trimerisation core bound with ssDNA (∼55 kDa) at ∼4.7 Å resolution and a dimeric RPA assembly on ssDNA. FRET-based solution studies reveal dynamic rearrangements of DBDs during coordinated RPA binding and this activity is regulated by phosphorylation at S178 in RPA70. We present a structural model on how dynamic DBDs promote the cooperative assembly of multiple RPAs on long ssDNA.
复制蛋白 A（RPA）是主要的真核生物单链 DNA 结合蛋白，可结合暴露的单链 DNA 以保护其免受核酸酶的侵害，参与多种核酸代谢过程，并协调其他重要因子的募集。RPA 是一种异三聚体，可覆盖长链单链 DNA（ssDNA）。在组装过程中，RPA 亚基及其 DNA 结合结构域（DBD）的精确分子结构尚不清楚。我们利用冷冻电镜获得了与 ssDNA（约 55 kDa）结合的 RPA 三聚体核心的 3D 重建结构，分辨率约为 4.7 Å，以及在 ssDNA 上的二聚体 RPA 组装。基于荧光共振能量转移（FRET）的溶液研究揭示了 DBD 在协调的 RPA 结合过程中的动态重排，这种活性受 RPA70 中 S178 位的磷酸化调节。我们提出了一个结构模型，说明动态 DBD 如何促进多个 RPA 在长 ssDNA 上的协同组装。
