Abstract
Replication factor C (RFC) is a five-subunit protein complex required for coordinate leading and lagging strand DNA synthesis during S phase and DNA repair in eukaryotic cells. It functions to load the proliferating cell nuclear antigen (PCNA), a processivity factor for polymerases delta and epsilon, onto primed DNA templates. This process, which is ATP-dependent, is carried out by 1) recognition of the primer terminus by RFC () binding to and disruption of the PCNA trimer, and then 3) topologically linking the PCNA to the DNA. In this report, we describe the purification and properties of recombinant human RFC expressed in Sf9 cells from baculovirus expression vectors. Like native RFC derived from 293 cells, recombinant RFC was found to support SV40 DNA synthesis and polymerase delta DNA synthesis in vitro and to possess an ATPase activity that was highly stimulated by DNA and further augmented by PCNA. Assembly of RFC was observed to involve distinct subunit interactions in which both the 36- and 38-kDa subunits interacted with the 37-kDa subunit, and the 40-kDa subunit interacted with the 36-kDa subunit-37-kDa subunit subcomplex. The 140-kDa subunit was found to require interactions primarily with the 38- and 40-kDa subunits for incorporation into the complex. In addition, a stable subcomplex lacking the 140-kDa subunit, although defective for DNA replication, was found to possess DNA-dependent ATPase activity that was not responsive to the addition of PCNA.
Highlights
In contrast to the function of RFC in DNA replication, its role in DNA repair is less clear
Expression of the large subunit was confirmed by immunoblotting, and expression of the 38-kDa subunit was confirmed by analysis of protein synthesis by [35S]methionine labeling of the cells 42 h postinfection, when viral mRNAs were predominantly translated (Fig. 1B, lane 5)
Using the HA epitope variant of the 140-kDa subunit (HA140kDa), we observed co-purification of the 40, 38, 37, and 36-kDa subunits with the HA-140kDa subunit on a column consisting of the monoclonal antibody 12CA5 linked to protein A-Sepharose (Fig. 2A, lanes labeled E1 and E2), suggesting that the RFC complex was reconstituted in the infected cells
Summary
In contrast to the function of RFC in DNA replication, its role in DNA repair is less clear. Mutations in the S. cerevisiae gene encoding the large subunit of RFC (cdc44) have been shown to render the cell sensitive to exposure to the alkylating agent methylmethane sulfonate and UV radiation, but not ␥ irradiation, suggesting a role for RFC in the base excision and nucleotide excision repair pathways [8]. Consistent with this hypothesis, mutations in the pol gene encoding PCNA have been found to suppress the DNA repair defect in cdc mutants [8, 13]. To investigate the many interesting structurefunction relationships in human RFC, we have reconstituted in active form a recombinant human RFC in Sf9 cells and characterized the subunit requirements for complex assembly and activity
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