Replication protein A interactions with DNA. 1. Functions of the DNA-binding and zinc-finger domains of the 70-kDa subunit

Human replication protein A (RPA) is a multiple subunit single-stranded DNA -binding protein that is required for multiple processes in cellular DNA me tabolism. This complex, composed of subunits of 70, 32, and 14 kDa, binds t o single-stranded DNA (ssDNA) with high affinity and participates in multip le protein-protein interactions. The 70-kDa subunit of RPA is known to be c omposed of multiple domains: an N-terminal domain that participates in prot ein interactions, a central DNA-binding domain (composed of two copies of a ssDNA-binding motif), a putative (C-X-2-C-X-13-C-X-2-C) zinc finger, and a C-terminal intersubunit interaction domain. A series of mutant forms of RP A were used to elucidate the roles of these domains in RPA function. The ce ntral DNA-binding domain was necessary and sufficient for interactions with ssDNA; however, adjacent sequences, including the zinc-finger domain and p art of the N-terminal domain, were needed for optimal ssDNA-binding activit y. The role of aromatic residues in RPA-DNA interactions was examined. Muta tion of any one of the four aromatic residues shown to interact with ssDNA had minimal effects on RPA activity, indicating that individually these res idues are not critical for RPA activity. Mutation of the zinc-finger domain altered the structure of the RPA complex, reduced ssDNA-binding activity, and eliminated activity in DNA replication.