Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells

Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12896-901. doi: 10.1073/pnas.93.23.12896.


Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase delta or epsilon. Here we report the reconstitution of the RFC complex from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified baculovirus-produced RFC appears to contain equimolar levels of each subunit and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase delta-catalyzed PCNA-dependent DNA chain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (iii) binding preferentially to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Animals
  • Baculoviridae
  • Cell Line
  • DNA Primers
  • DNA Replication*
  • DNA-Binding Proteins / biosynthesis*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / isolation & purification
  • HeLa Cells
  • Homeodomain Proteins*
  • Humans
  • Kinetics
  • Macromolecular Substances
  • Minor Histocompatibility Antigens
  • Proliferating Cell Nuclear Antigen / analysis
  • Proto-Oncogene Proteins c-bcl-2*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Replication Protein C
  • Repressor Proteins*
  • Saccharomyces cerevisiae Proteins*
  • Spodoptera
  • Transfection


  • BCL2-related protein A1
  • DNA Primers
  • DNA-Binding Proteins
  • Homeodomain Proteins
  • MATA1 protein, S cerevisiae
  • Macromolecular Substances
  • Minor Histocompatibility Antigens
  • Proliferating Cell Nuclear Antigen
  • Proto-Oncogene Proteins c-bcl-2
  • RFC2 protein, human
  • RFC3 protein, human
  • RFC4 protein, human
  • Recombinant Proteins
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • Adenosine Triphosphatases
  • Replication Protein C