THE EVOLUTIONARILY CONSERVED ZINC-FINGER MOTIF IN THE LARGEST SUBUNITOF HUMAN REPLICATION PROTEIN-A IS REQUIRED FOR DNA-REPLICATION AND MISMATCH REPAIR BUT NOT FOR NUCLEOTIDE EXCISION-REPAIR

The largest subunit of the replication protein A (RPA) contains an evo lutionarily conserved zinc finger motif that lies outside of the domai ns required for binding to single-stranded DNA or forming the RPA holo complex. In previous studies, we showed that a point mutation in this motif (RPA(m)) cannot support SV40 DNA replication. We have now invest igated the role of this motif in several steps of DNA replication and in two DNA repair pathways. RPA(m) associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stim ulates DNA polymerases alpha and delta, and supports the formation of the initial short Okazaki fragments. However, the synthesis of a leadi ng strand and later Okazaki fragments is impaired. In contrast, RPA(m) can function well during the incision step of nucleotide excision rep air and in a full repair synthesis reaction, with either UV-damaged or cisplatin-adducted DNA. Two deletion mutants of the Rpa1 subunit (eli minating amino acids 1-278 or 222-411) were not functional in nucleoti de excision repair. We report for the first time that wild type RPA is required for a mismatch repair reaction in vitro. Neither the deletio n mutants nor RPA(m) can support this reaction. Therefore, the zinc fi nger of the largest subunit of RPA is required for a function that is essential for DNA replication and mismatch repair but not for nucleoti de excision repair.