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Recombinant replication protein A: expression, complex formation, and functional characterization

L A Henricksen1, C B Umbricht, M S Wold

  • 1Department of Biochemistry, University of Iowa, Iowa City 52242.

The Journal of Biological Chemistry
|April 15, 1994
PubMed
Summary
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Researchers successfully produced functional recombinant human replication protein A (RPA) in E. coli. This essential DNA-binding protein complex supports SV40 DNA replication, paving the way for further biochemical studies.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Virology

Background:

  • Replication protein A (RPA) is a crucial multisubunit protein essential for DNA replication.
  • Human RPA consists of 70, 32, and 14 kDa subunits.
  • SV40 DNA replication requires RPA activity.

Purpose of the Study:

  • To express and characterize human RPA subunits and their complexes in E. coli.
  • To generate functional recombinant human RPA for biochemical analysis.
  • To investigate the assembly and properties of RPA.

Main Methods:

  • Expression of individual and combined human RPA subunit cDNAs in E. coli.
  • Purification and characterization of RPA subcomplexes and the complete RPA complex.
  • In vitro assays to assess DNA binding and replication activity.

Related Experiment Videos

  • Phosphorylation assays of RPA under replication conditions.

    • Main Results:

    • Individual RPA subunits expressed in E. coli were largely insoluble or aggregated.
    • A soluble complex formed only between the 32 and 14 kDa subunits (32-14 kDa subcomplex).
    • Coexpression of all three subunits yielded a soluble, functional recombinant human RPA complex.
    • Recombinant human RPA exhibited properties similar to native RPA, including DNA binding and support of SV40 DNA replication.
    • Recombinant RPA was phosphorylated under replication conditions.

    Conclusions:

    • The 32-14 kDa subcomplex may serve as a precursor for RPA assembly.
    • Recombinant human RPA produced in E. coli is biochemically similar to native RPA.
    • This recombinant RPA is suitable for in vitro studies of DNA replication mechanisms.