Modification Reactivity Analysis of Human Replication Protein a in Biologically Important States

Human Replication Protein A (RPA) is a heterotrimeric protein consisting of 70, 32, and 14 kDa subunits. RPA is the predominant single stranded DNA binding protein within the cell. It is involved in all forms of the DNA metabolic pathways, including but not limited to, replication, recombination, damage repair, as well as cell cycle and DNA check point signaling. RPA is phosphorylated (pRPA) during G1-S phase and is dephosphorylated during M phase. Further, RPA is hyperphosphorylated during DNA damage. Through the use of x-ray crystallography and nuclear magnetic resonance, researchers have proposed models and structures based on truncated portions of the protein. Currently, there are no x-ray crystallographic or NMR models for the full RPA heterotrimer. Our lab, using chemical modification reactivity analysis data (MRAN) and simulated annealing, has a proposed model for the complete structure of native RPA unbound to ssDNA. To further refine models for the complete structure of RPA, we have used a series of experiments in which the structure of RPA was probed via Chemical Modification Reactivity Analysis (MRAN). Specifically, lysines within RPA were probed by sulfo-NHS-acetate and aspartates and glutamates were probed with N-(3-dimethylaminopropyl)-N\u27-ethylcarbodiimide hydrochloride (EDC) chemical reagents under conditions in which RPA was in native, phoshphorylated, single strand DNA (ssDNA) bound, and phosphorylated ssDNA bound states to determine the structural changes that result from different binding and phosphorylation states. Chemical modification and exhaustive proteolysis were used in conjunction with MALDI-TOF mass spectrometry to determine local and global structural changes that occur within the protein under each specific reaction condition. Through the use of photochemical crosslinks between RPA and dT30 ssDNA, the sites and specific amino acids involved in binding between RPA and ssDNA were mapped. All DNA binding domains (DBDs) of the heterotrimer were shown to interact with the ssDNA including the 14 kDa subunit (DBD E). The interaction between DBDs A, B, C, D, and F coincided with reports of the interaction between RPA and ssDNA. Our data for the interaction between DBD E of the 14 kDa subunit and ssDNA provides a strong argument that the 14kDa subunit is involved in at least one mechanism of ssDNA binding within the cell. MRAN data further suggests that the modular rearrangement of RPA is necessary, and that RPA goes from a compact conformation with DBDs A & B docked to the trimerization core in the ligand-free state to an extended conformation in which DBDs A & B are extended by a tethered region away from the trimerization core of RPA when bound to ssDNA. Further, the structural analysis of RPA and pRPA through chemical modification reactivity analyses in both apo and ssDNA bound states provides greater detail of the local conformational changes that the heterotrimer undergoes between these states. This data suggests that RPA and pRPA are similar and that the molecule undergoes specific local changes due to changes in electrostatic and hydrophobic potential of specific amino acids within each domain. It further demonstrates that the local changes in surface accessibility do not affect global structure of the domains, or of the molecule as a whole, but rather represents minor changes that influence specific amino acid-ligand interactions. The data suggest that RPA and pRPA undergo a significant rearrangement when bound to ssDNA. The dissociation of DBDs A & B from the trimerization core represents a significant change in structure from the ssDNA free state. Evaluation of these changes in relation to proposed structures for RPA allows for targeted adjustments to the proposed models to better fit experimental data and gives a more accurate representation of the structure in solution under each reaction condition