Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells
- 12 November 1996
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 93 (23) , 12896-12901
- https://doi.org/10.1073/pnas.93.23.12896
Abstract
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 δ or ɛ. 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 δ-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.Keywords
This publication has 47 references indexed in Scilit:
- DNA EXCISION REPAIRAnnual Review of Biochemistry, 1996
- A Molecular Switch in a Replication Machine Defined by an Internal Competition for Protein RingsCell, 1996
- Clamp loading, unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human, E. coli and T4 replicasesGenes to Cells, 1996
- Escherichia coli expression vectors containing a protein kinase recognition motif, His6-tag and hemagglutinin epitopeGene, 1995
- Calf 5′ to 3′ Exo/Endonuclease Must Slide from a 5′ End of the Substrate to Perform Structure-specific CleavagePublished by Elsevier ,1995
- Mammalian DNA nucleotide excision repair reconstituted with purified protein componentsCell, 1995
- Sliding Clamps of DNA PolymerasesJournal of Molecular Biology, 1993
- Cloning and Expression of a Novel Human DNA Binding Protein, PO-GABiochemical and Biophysical Research Communications, 1993
- A Transcriptional Enhancer Whose Function Imposes a Requirement That Proteins Track Along DNAScience, 1992
- Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4.Proceedings of the National Academy of Sciences, 1990