FUNCTIONAL DOMAINS OF THE 70-KILODALTON SUBUNIT OF HUMAN REPLICATION PROTEIN-A

Human replication protein A (RPA) is a single-stranded DNA-binding pro tein that is composed of submits of 70, 32, and 14 kDa. This heterotri meric complex is required for multiple processes in DNA metabolism inc luding DNA replication, DNA repair, and recombination. Previous studie s have suggested that the 616 amino acid: 70-kDa subunit of RPA (RPA70 ) is composed of multiple structural/functional domains. We used a ser ies of N-terminal deletions of RPA70 to define the boundaries of these domains and elucidate their functions. Mutant RPA complexes missing r esidues 1-168 of RPA70 bound ssDNA with high affinity and supported SV 40 replication in vitro. In contrast, deletions extending beyond resid ue 168 showed a decreased affinity for ssDNA and were inactive in SV40 DNA replication. When residues 1-381 were deleted, the resulting trun cated 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. T hese data indicate that RPA70 is composed of three functional domains: an N-terminal domain that is not required for ssDNA binding or SV40 r eplication, a central DNA-binding domain, and a C-terminal domain that is essential for subunit interactions. For all mutant complexes exami ned, both phosphorylation of the 32-kDa subunit of RPA and the ability to support T antigen-dependent, origin-dependent DNA unwinding correl ated with ssDNA binding activity.