The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding
The weak interdomain coupling observed in the 70 kDa subunit of human replication protein A is unaffected by ssDNA binding
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DOI：
10.1093/nar/29.15.3270
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发表时间：
2001-08-01
影响因子：
14.9
通讯作者：
Lowry, DF
中科院分区：
文献类型：
--
作者：
Daughdrill, GW;Ackerman, J;Lowry, DF
复制蛋白A（RPA）是一种异源三聚体多功能蛋白，可结合单链DNA（ssDNA），对真核生物DNA代谢至关重要。我们用异相NMR方法研究了人RPA 70亚基片段（hRPA 70）的结构域相互作用和ssDNA结合。该片段含有一个N-末端结构域（NTD），它对hRPA 70-蛋白质相互作用很重要，通过柔性接头（hRPA 70（1-326））连接到ssDNA结合结构域（SSB 1）。使用酰胺H-1和N-15化学位移的相关性分析来比较hRPA 70（1-326）中NTD和SSB 1的结构与对应于各个结构域的两个较小片段。高相关系数验证了NTD和SSB 1在hRPA 70（1-326）中保持其结构，表明弱的域间偶联。通过比较hRPA 70（1-326）和含有NTD和柔性接头的较小hRPA 70片段之一（hRPA 70（1-168））的横向弛豫速率，也表明了弱的结构域间偶联。我们还检查了在添加三种不同ssDNA底物后hRPA 70（1-326）的结构。这些底物中的每一种在NTD和SSB 1中都诱导特定的酰胺H-1和/或N-15化学位移变化。NTD和SSB 1具有相似的拓扑结构，导致ssDNA结合诱导NTD观察到的化学位移变化的可能性。为了检验这一假设，我们监测了添加ssDNA后hRPA 70（1-168）的酰胺H-1和N-15化学位移变化。对于hRPA 70（1-168）和hRPA 70（1-326）中的NTD，观察到相同的酰胺H-1和N-15化学位移变化。具有最大酰胺H-1和/或N-15化学位移变化的NTD残基定位于对hRPA 70-蛋白质相互作用重要的碱性裂缝。基于这种关系，以及其他可用的数据，我们提出了一个模型，其中NTD和ssDNA之间的结合干扰hRPA 70-蛋白质相互作用。
Replication protein A (RPA) is a heterotrimeric, multifunctional protein that binds single-stranded DNA (ssDNA) and is essential for eukaryotic DNA metabolism. Using heteronuclear NMR methods we have investigated the domain interactions and ssDNA binding of a fragment from the 70 kDa subunit of human RPA (hRPA70). This fragment contains an N-terminal domain (NTD), which is important for hRPA70-protein interactions, connected to a ssDNA-binding domain (SSB1) by a flexible linker (hRPA70(1-326)). Correlation analysis of the amide H-1 and N-15 chemical shifts was used to compare the structure of the NTD and SSB1 in hRPA70(1-326) with two smaller fragments that corresponded to the individual domains. High correlation coefficients verified that the NTD and SSB1 maintained their structures in hRPA70(1-326), indicating weak interdomain coupling. Weak interdomain coupling was also suggested by a comparison of the transverse relaxation rates for hRPA70(1-326) and one of the smaller hRPA70 fragments containing the NTD and the flexible linker (hRPA70(1-168)). We also examined the structure of hRPA70(1-326) after addition of three different ssDNA substrates. Each of these substrates induced specific amide H-1 and/or N-15 chemical shift changes in both the NTD and SSB1. The NTD and SSB1 have similar topologies, leading to the possibility that ssDNA binding induced the chemical shift changes observed for the NTD. To test this hypothesis we monitored the amide H-1 and N-15 chemical shift changes of hRPA70(1-168) after addition of ssDNA. The same amide H-1 and N-15 chemical shift changes were observed for the NTD in hRPA70(1-168) and hRPA70(1-326). The NTD residues with the largest amide H-1 and/or N-15 chemical shift changes were localized to a basic cleft that is important for hRPA70-protein interactions. Based on this relationship, and other available data, we propose a model where binding between the NTD and ssDNA interferes with hRPA70-protein interactions.