STRUCTURAL-ANALYSIS OF HUMAN REPLICATION PROTEIN-A - MAPPING FUNCTIONAL DOMAINS OF THE 70-KDA SUBUNIT

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-bi nding protein that is essential for DNA metabolism. Human RPA is compo sed of subunits of 70, 32, and 14 kDa with intrinsic DNA-binding activ ity localized to the 616-amino acid, 70-kDa subunit (RPA70). We have m ade a series of C-terminal deletions to map the functional domains of RPA70. Deletion of the C terminus resulted in polypeptides that were s ignificantly more soluble than RPA70 but were unable to form stable co mplexes with the other two subunits of RPA. These data suggest that th e C-terminal region of RPA70 may be important for complex formation. T he DNA-binding do main was localized to a region of RPA70 between resi dues 1 and 441. A mutant containing residues 1-441 bound oligonucleoti des with an intrinsic affinity close to wild-type RPA complex. This mu tant also appeared to bind with reduced cooperativity. We conclude tha t the C terminus of RPA70 and the 32- and 14-kDa subunits are not invo lved directly with interactions with DNA but may have a role in cooper ativity of RPA binding. RPA70 deletion mutants were not able to suppor t DNA replication even in the presence of a complex of the 32- and 14- kDa subunits, suggesting that the heterotrimeric complex is essential for DNA replication. The putative zinc finger in the C terminus of RPA 70 is not required for single-stranded DNA-binding activity.