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-binding 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 RPA14, respectively). We examined the structure of both wild-type and mutant forms of human RPA by mapping sites sensitive to proteolytic cleavage. For all three subunits, only a subset of the possible protease cleavage sites was sensitive to digestion. RPA70 was cleaved into multiple fragments of defined lengths. RPA32 was cleaved rapidly to a approximately 28-kDa polypeptide containing the C-terminus that was partially resistant to further digestion. RPA14 was refractory to digestion under the conditions used in these studies. The digestion properties of a complex of RPA32 and RPA14 were similar to those of the native heterotrimeric RPA complex, indicating that the structure of these subunits is similar in both complexes. Epitopes recognized by monoclonal antibodies to RPA70 were mapped, and this information was used to determine the position of individual cleavage events. These studies suggest that RPA70 is composed of at least two structural domains: an approximately 18-kDa N-terminal domain and a approximately 52-kDa C-terminal domain. The N-terminus of RPA70 was not required for single-stranded DNA-binding activity. Multiple changes in the digestion pattern were observed when RPA bound single-stranded DNA: degradation of the approximately 52-kDa domain of RPA70 was inhibited while proteolysis of RPA32 was stimulated. These data indicate that RPA undergoes a conformational change upon binding to single-stranded DNA.
Gomes
X V
XV
Department of Biochemistry, University of Iowa School of Medicine, Iowa City 52242-1109, USA.
Henricksen
L A
LA
Wold
M S
MS
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011487:Protein Conformation; D011994:Recombinant Proteins; D051716:Replication Protein A; D017384:Sequence Deletion
D000911:Antibodies, Monoclonal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A