Functional domains of the 70-kilodalton subunit of human replication protein A

Abstract

Human replication protein A (RPA) is a single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. This heterotrimeric complex is required for- multiple processes in DNA metabolism including DNA replication. DNA repair, trod recombination. Previous studies have suggested that the 616 amino acid, 70-kDa subunit of RPA (RPA70) is composed of multiple structural/functional domains. We used a series of N terminal deletions of RPA70 to define the boundaries of these domains and elucidate their functions. Mutant RPA complexes missing residues 1-168 of RPA70 bound ssDNA with high affinity and supported SV40 replication in vitro. In contrast, deletions extending beyond residue 168 showed a decreased affinity for ssDNA and were inactive in SV40 DNA replication. When residues 1-381 were deleted, the resulting truncated RPA70 was unable to bind ssDNA but still formed a stable complex with the 32- and 14-kDa subunits of RPA. Thus, the C-terminal domain of RPA70 is both necessary and sufficient for RPA complex formation. These data indicate that RPA70 is composed of three functional domains: an N-terminal domain that is not required for ssDNA binding or SV40 replication, a central DNA-binding domain and a C-terminal domain that is essential for subunit interactions. For all mutant complexes examined, both phosphorylation of the 32-kDa subunit of RPA and the ability to support T antigen-dependent, origin-dependent DNA unwinding correlated with ssDNA binding activity.