The Weak Interdomain Coupling Observed in the 70 kDa Subunit of Human Replication Protein A is Unaffected by ssDNA Bindin

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Replication protein A (RPA) is a heterotrimeric, multi-functional 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 (hRPA701–326). Correlation analysis of the amide 1H and 15N chemical shifts was used to compare the structure of the NTD and SSB1 in hRPA701–326 with two smaller fragments that corresponded to the individual domains. High correlation coefficients verified that the NTD and SSB1 maintained their structures in hRPA701–326, indicating weak interdomain coupling. Weak interdomain coupling was also suggested by a comparison of the transverse relaxation rates for hRPA701–326 and one of the smaller hRPA70 fragments containing the NTD and the flexible linker (hRPA701–168). We also examined the structure of hRPA701–326 after addition of three different ssDNA substrates. Each of these substrates induced specific amide 1H and/or 15N 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 1H and 15N chemical shift changes of hRPA701–168 after addition of ssDNA. The same amide 1H and 15N chemical shift changes were observed for the NTD in hRPA701–168 and hRPA701–326. The NTD residues with the largest amide 1H and/or 15N 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.

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Nucleic Acids Research, v. 29, issue 15, p. 3270-3276