IN-VITRO RECONSTITUTION OF HUMAN REPLICATION FACTOR-C FROM ITS 5 SUBUNITS

Replication factor C (RFC, also called Activator I) is part of the pro cessive eukaryotic DNA polymerase holoenzymes. The processive elongati on of DNA chains requires that DNA polymerases are tethered to templat e DNA at primer ends, In eukaryotes the ring-shaped homotrimeric prote in, proliferating cell nuclear antigen (PCNA), ensures tight template- polymerase interaction by encircling the DNA strand, Proliferating cel l nuclear antigen is loaded onto DNA through the action of RFC in an A TP-dependent reaction, Human RFC is a protein complex: consisting of f ive distinct subunits that migrate through SDS/polyacrylamide gels as protein bands of 140, 40, 38, 37, and 36 kDa, All five genes encoding the RFC subunits have been cloned and sequenced, A functionally identi cal RFC complex has been isolated from Saccharomyces cerevisiae and th e deduced amino acid sequences among the corresponding human and yeast subunits are homologous. Here we report the expression of the five cl oned human genes using an In vitro coupled transcription/translation s ystem and show that the gene products form a complex resembling native RFC that is active In supporting an RFC-dependent replication reactio n, Studies on the interactions between the five subunits suggest a coo perative mechanism in the assembly of the RFC complex, A three-subunit core complex, consisting of p36, p37, and p40, was identified and evi dence is presented that p38 is essential for the interaction between t his core complex and the large p140 subunit.