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 δ 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.