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Replication protein A (RPA) is the major eukaryotic single-strand DNA (ssDNA) binding protein. RPA is composed of three subunits, RPA1, RPA2 and RPA3. RPA is essential for replication, repair, recombination, and checkpoint activation, and is required for maintaining genome integrity. In the cell, RPA binds to ssDNA intermediates and ensures that the appropriate pathway correctly processes them. The ssDNA-binding activity of RPA is primarily mediated by two high-affinity domains in the RPA1 subunit. DNA binds to these domains by interacting with polar and aromatic residues in a DNA-binding cleft in each domain. The aromatic residues are highly conserved and when mutated cause a separation-of-function phenotype.
Mutation of the conserved aromatic residues in the high-affinity binding domains of RPA only modestly affected the affinity of RPA but these aromatic residue mutants were unable to support DNA repair while functioning in DNA replication. To understand the molecular basis of this phenotype, I have characterized the interactions of the aromatic mutants with different length ssDNAs and partial duplex DNA structures like those found in DNA repair. I also probed the conformations and dynamics of RPA-DNA complexes. My studies identified that there are at least two kinetic states when RPA binds to ssDNA that differ in their rate of dissociation from the DNA. I also showed that the aromatic residues are required for the stable binding to short ssDNA and contribute to the formation of the more long-lived state. My studies also showed that the more stable state is important for RPA in melting secondary DNA structure. We conclude that melting activity and/or stable binding by RPA is required for DNA repair but dispensable for DNA replication. These studies enhance our understating of molecular interactions between RPA and DNA that contribute to different cellular functions.
The kinetic states in RPA could reflect changes in domain interactions or changes in conformation of the RPA-DNA complex. To try to understand the molecular basis of the different kinetic states, I used single molecule FRET analysis to characterize the spatial location of RPA domains and conformational dynamics in RPA-DNA complex. My studies showed RPA binds different locations along ssDNA and that generally RPA does not undergo global changes in conformation when bound to ssDNA. However, with a subset of label locations, some RPA-DNA complexes showed rare changes in conformation. These observations were most consistent with partial microscopic dissociation (domains of RPA partially dissociate from DNA, but has not yet equilibrated with the surrounding solution) of domains of RPA near the 3’ end of the complex and interactions of the flexible N-terminal, regulatory domain of RPA with the free DNA. My data suggests that the microscopic dissociation can occur without affecting the global structure of the RPA-DNA complex.
These studies illustrate that different DNA metabolic pathways require different types of RPA-DNA complexes and that high affinity binding is not sufficient for all RPA functions. Specifically, my studies showed that DNA repair pathways require different ssDNA interactions. This suggests that modulation of the binding of individual domains and/or inter-domain interactions regulates the properties of the RPA-DNA complex and, in turn, that this could direct ssDNA intermediates into different pathways for processing. Together, my studies highlight the importance of dynamics in RPA binding to properly maintain the integrity of the genome.