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Related Experiment Videos

Assembly of functional replication factor C expressed using recombinant baculoviruses

V N Podust1, E Fanning

  • 1Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

The Journal of Biological Chemistry
|March 7, 1997
PubMed
Summary

Researchers created a functional human Replication Factor C (RF-C) complex using baculovirus expression. This essential protein complex is crucial for DNA replication and repair, and this study outlines its assembly process.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Replication factor C (RF-C) is a vital five-subunit eukaryotic protein complex.
  • RF-C plays critical roles in DNA replication and DNA repair pathways.

Purpose of the Study:

  • To develop an accessible method for producing functional human RF-C for research.
  • To elucidate the assembly process of the human RF-C complex.

Main Methods:

  • Cloning cDNAs of all five RF-C subunits into baculoviruses for expression in insect cells.
  • Co-infection of insect cells to express individual subunits, including His-tagged variants.
  • Assessing RF-C activity through DNA polymerase delta stimulation and PCNA complex formation assays.
  • Utilizing co-purification experiments to determine subunit assembly order.

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Main Results:

  • Co-expression of all five subunits was necessary to reconstitute active RF-C.
  • Functional RF-C containing His-tagged subunits (p40, p37, p36) was successfully isolated.
  • A model for RF-C assembly was proposed, starting with p37/p36, followed by p40/p38, then the fourth small subunit, and finally p140.

Conclusions:

  • The study successfully produced functional human RF-C using a baculovirus co-expression system.
  • The findings provide insights into the sequential assembly of the five RF-C subunits.
  • This work facilitates further biochemical and genetic studies of RF-C's function in DNA metabolism.