RECONSTITUTION OF HUMAN REPLICATION FACTOR-C FROM ITS 5 SUBUNITS IN BACULOVIRUS-INFECTED INSECT CELLS

Human replication factor C (RFC, also called activator I) is a five-su bunit protein complex (p140, p10, p38, p37, and p36) required for prol iferating cell nuclear antigen (PCNA)-dependent processive DNA synthes is catalyzed by DNA polymerase delta or epsilon. Here we report the re constitution of the RFC comples from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified bacul ovirus-produced RFC appears to contain equimolar levels of each subuni t and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase delta-catalyzed PCNA-dependent DNA c hain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (ii i) binding preferentiaIly to DNA primer ends; and (iv) catalytically l oading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.