Assembly of functional replication factor C expressed using recombinant baculoviruses.
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.
Podust
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Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Fanning
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D016367:Baculoviridae; D004258:DNA Polymerase III; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D018398:Homeodomain Proteins; D006801:Humans; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D008970:Molecular Weight; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
C096816:BCL2-related protein A1; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; C497561:RFC2 protein, human; C497562:RFC3 protein, human; C497563:RFC4 protein, human; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D051818:Replication Protein C