Domain-based structural studies of Replication Protein A: analysis of an RPA32N phospho-mimic mutant and the role of RPA70N in binding ssDNA

Abstract

Replication Protein A (RPA) is the primary eukaryotic ssDNA binding protein utilized in preventing secondary structure formation and re-annealing of unwound DNA strands, thereby controlling access to DNA templates in diverse DNA transactions in the cell. Importantly, RPA serves as a scaffold for the assembly/disassembly of DNA processing machinery during normal cell cycle conditions and when DNA damage is encountered. The question of how RPA recognizes replication versus repair proteins, or those of other pathways remains obscure. However it is known that hyperphosphorylation of RPA signals recognition of DNA damage. RPA is a heterotrimer composed of three subunits (RPA70, RPA32, RPA14) that contain seven globular domains (70N, 70A, 70B, 70C, 32D, 32C) and one unstructured domain (32N) harboring all phosphorylation sites observed in DNA damage recognition. The domains are connected by flexible linkers that enable substantial inter-domain motion essential to RPA function. Small angle X-ray scattering (SAXS) experiments on two multi-domain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains, but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA bound state and therefore freely available to serve as a protein recruitment module. Nuclear magnetic resonance (NMR) experiments on RPA32N and an RPA32N phospho-mimic mutant (RPA32N-D8) were used to systematically examine the effect of hyperphosphorylation on interactions of RPA32N with other RPA domains. Multiple weak interactions between RPA32N-D8 and the 70N, 70A and 70B domains were observed. Control experiments demonstrated there were no interactions with the 32D, 32C and 14 domains. The data were interpreted within a framework in which RPA becomes more compact upon hyperphosphorylation. Such a change in the structural dynamics of RPA would be expected to influence its protein interaction network as part of the switch in activity upon damage of DNA.