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

   


Authors: Cai, J.; Uhlmann, F.; Gibbs, E.; Flores-Rozas, H.; Lee, C. G.; Phillips, B.; Finkelstein, J.; Yao, N.; O'Donnell, M.; Hurwitz, J.
Article Title: Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells
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 ε. 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: dna-binding proteins; conference paper; dna polymerase; dna replication; dna synthesis; animals; protein assembly; cell protein; cell line; homeodomain proteins; hela cells; transfection; dna; kinetics; eukaryota; recombinant proteins; spodoptera; saccharomyces cerevisiae proteins; dna primers; adenosine triphosphate; cycline; protein subunit; replication protein c; dna binding; repressor proteins; adenosine triphosphatases; proto-oncogene proteins c-bcl-2; biological factor; macromolecular substances; proliferating cell nuclear antigen; insect; baculovirus; baculoviridae; unidentified baculovirus; insecta; humans; priority journal; dna polymerase δ; activator i; eukaryotic dna replication
Journal Title: Proceedings of the National Academy of Sciences of the United States of America
Volume: 93
Issue: 23
ISSN: 0027-8424
Publisher: National Academy of Sciences  
Date Published: 1996-11-12
Start Page: 12896
End Page: 12901
Language: English
DOI: 10.1073/pnas.93.23.12896
PUBMED: 8917516
PROVIDER: scopus
PMCID: PMC24017
DOI/URL:

https://www.scopus.com/inward/record.uri?eid=2-s2.0-10544250652&doi=10.1073%2fpnas.93.23.12896&partnerID=40&md5=03658eab5c24377a295d2274ce1b399d
Notes: Conference Paper -- Export Date: 22 November 2017 -- Source: Scopus
Altmetric
What is Altmetric?
Citation Impact
What is Dimensions Citation Badge?
BMJ Impact Analytics
MSK Authors
  1. Jerard Hurwitz
    206 Hurwitz
  2. Barbara Phillips
    6 Phillips
  3. Chee-Gun Lee
    11 Lee
  4. Emma Gibbs
    14 Gibbs
Related MSK Work