title: The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction.
abstract: Replication protein A (RPA) is a heterotrimeric (subunits of 70, 32, and 14 kDa) single-stranded DNA-binding protein that is required for DNA replication, recombination, and repair. The 40-residue N-terminal domain of the 32-kDa subunit of RPA (RPA32) becomes phosphorylated during S-phase and after DNA damage. Recently it has been shown that phosphorylation or the addition of negative charges to this N-terminal phosphorylation domain modulates RPA-protein interactions and increases cell sensitivity to DNA damage. We found that addition of multiple negative charges to the N-terminal phosphorylation domain also caused a significant decrease in the ability of a mutant form of RPA to destabilize double-stranded (ds) DNA. Kinetic studies suggested that the addition of negative charges to the N-terminal phosphorylation domain caused defects in both complex formation (nucleation) and subsequent destabilization of dsDNA by RPA. We conclude that the N-terminal phosphorylation domain modulates RPA interactions with dsDNA. Similar changes in DNA interactions were observed with a mutant form of RPA in which the N-terminal domain of the 70-kDa subunit was deleted. This suggested a functional link between the N-terminal domains of the 70- and 32-kDa subunits of RPA. NMR experiments provided evidence for a direct interaction between the N-terminal domain of the 70-kDa subunit and the negatively charged N-terminal phosphorylation domain of RPA32. These findings suggest that phosphorylation causes a conformational change in the RPA complex that regulates RPA function.
authors.affiliation: Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242-1109, USA.
authors.forename: Sara K
authors.initials: SK
authors.lastname: Binz
authors.forename: Ye
authors.initials: Y
authors.lastname: Lao
authors.forename: David F
authors.initials: DF
authors.lastname: Lowry
authors.forename: Marc S
authors.initials: MS
authors.lastname: Wold
mesh_terms: D000818:Animals; D004249:DNA Damage; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D051716:Replication Protein A; D017384:Sequence Deletion
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D051716:Replication Protein A