ROLE OF THE 70-KDA SUBUNIT OF HUMAN REPLICATION PROTEIN-A .1. SINGLE-STRANDED-DNA BINDING-ACTIVITY, BUT NOT POLYMERASE STIMULATORY ACTIVITY, IS REQUIRED FOR DNA-REPLICATION

Replication protein A (RPA), also known as human single-stranded DNA-b inding protein, is a three-subunit protein complex with multiple funct ions. Here, we investigated the role of the 70-kDa RPA subunit (p70) i n DNA replication, by generating a series of deletion mutants. Mutant p70, which lacked 50 amino acids at the C-terminus, failed to interact with the 11-kDa RPA subunit (p11) and, when deleted further at the C terminus, was unable to interact with either the 34-kDa subunit (p54) or with pll, suggesting that p70 directly interacts with both p34 and pll. Studies with purified RPA mutants indicated that deletions at the N-terminal domain of p70 had very little effect on RPA's single-stran ded DNA (ssDNA) binding activity, whereas deletion of amino acids 169- 246 significantly weakened the DNA binding ability of RPA. By deleting amino acids 296-373 or 374-458, we totally abolished p70's ssDNA bind ing activity, suggesting that multiple p70 domains are involved in DNA binding. Two p70 domains, the N-terminal domain and the DNA binding d omain, were required to stimulate DNA polymerase (pol) alpha, yet the DNA binding domain alone supported pol delta activity. Interestingly, RPA containing p70 with a zinc-finger domain deletion retained its DNA binding activity, but inhibited pol alpha and delta activity. RPA tha t lacked ssDNA binding activity failed to support simian virus 40 (SV4 0) DNA replication in vitro, whereas mutant RPA that lacked pol alpha stimulatory activity (including the zinc-finger p70 mutant) functioned normally. We conclude that RPA's DNA binding activity, but not its po l alpha stimulatory activity, is required for DNA replication.