Assembly of functional replication factor C expressed using recombinant baculoviruses.

ABSTRACT

Replication factor C (RF-C), a complex of five subunits, is an essential eukaryotic protein involved in both DNA replication and DNA repair. To generate an easily accessible source of human RF-C for biochemical and genetic studies, we cloned the cDNAs of all five subunits into baculoviruses so that each subunit could be expressed both as a non-fused polypeptide and as an N-terminal His-tagged fusion (-his). Co-infection of insect cells with five baculoviruses encoding individual RF-C subunits (p140, p40, p38, p37, and p36) yielded a protein preparation active in two assays characteristic for authentic RF-C stimulation of DNA polymerase delta DNA synthesis on singly primed single-stranded DNA template and formation of a complex of proliferating cell nuclear antigen with circular double-stranded DNA. Functional recombinant RF-C containing p40-his, p37-his, or p36-his was isolated using affinity resin. Active RF-C was reconstituted only by co-expression of all five subunits. A model for assembly of RF-C from individual subunits was derived from co-purification experiments performed with various combinations of His-tagged and non-fused subunits expressed by co-infection of insect cells with recombinant baculoviruses. p37 and p36 are proposed to form the first intermediate, which, upon addition of either p40 or p38, generates stable tertiary complexes p40.p37.p36 and p38.p37.p36. The remaining fourth small subunit binds to the tertiary complex to form a quaternary complex p40.p38. p37.p36. Large subunit p140 binds last to form the five-subunit protein.