PROTEOLYTIC MAPPING OF HUMAN REPLICATION PROTEIN-A - EVIDENCE FOR MULTIPLE STRUCTURAL DOMAINS AND A CONFORMATIONAL CHANGE UPON INTERACTION WITH SINGLE-STRANDED-DNA

Replication protein A (RPA) is multisubunit single-stranded DNA-bindin g protein required for multiple processes in DNA metabolism including DNA replication, DNA repair, and recombination. Human RPA is a stable complex of three subunits of 70, 32, and 14 kDa (RPA70, RPA32, and RPA 14, respectively). We examined the structure of both wild-type and mut ant forms of human RPA by mapping sites sensitive to proteolytic cleav age. For all three subunits, only a subset of the possible protease cl eavage sites was sensitive to digestion. RPA70 was cleaved into multip le fragments of defined lengths. RPA32 was cleaved rapidly to a simila r to 28-kDa polypeptide containing the C-terminus that was partially r esistant to further digestion. RPA14 was refractory to digestion under the conditions used in these studies. The digestion properties of a c omplex of RPA32 and RPA14 were similar to those of the native heterotr imeric RPA complex, indicating that the structure of these subunits is similar in both complexes. Epitopes recognized by monoclonal antibodi es to RPA70 were mapped, and this information was used to determine th e position of individual cleavage events. These studies suggest that R PA70 is composed of at least two structural domains: an similar to 18- kDa N-terminal domain and a similar to 52-kDa C-terminal domain. The N -terminus of RPA70 was not required for single-stranded DNA-binding ac tivity. Multiple changes in the digestion pattern were observed when R PA bound single-stranded DNA: degradation of the similar to 52-kDa dom ain of RPA70 was inhibited while proteolysis of RPA32 was stimulated. These data indicate that RPA undergoes a conformational change upon bi nding to single-stranded DNA.