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Further characterization of the human cell multiprotein DNA replication complex

N Applegren1, R J Hickey, A M Kleinschmidt

  • 1Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

Journal of Cellular Biochemistry
|September 1, 1995
PubMed
Summary

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Human cells contain a multiprotein complex (MRC) essential for DNA replication. This complex includes DNA polymerase and other key replication proteins, supporting origin-specific DNA synthesis in vitro.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • Growing evidence suggests multiprotein complexes are crucial for DNA replication.
  • Previous studies identified a replication-competent multiprotein DNA polymerase complex in human (HeLa) cell extracts.

Purpose of the Study:

  • To further characterize the human cell replication-competent multiprotein DNA polymerase complex, designated MRC.
  • To identify additional proteins co-purifying with the MRC and investigate its functional capabilities.

Main Methods:

  • Purification of the MRC using Q-Sepharose chromatography and glycerol gradient sedimentation.
  • Identification of co-purifying proteins using biochemical assays.
  • Assessment of DNA replication requirements, initiation kinetics, and minichromosome utilization.

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Main Results:

  • Several additional DNA replication proteins, including DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A, were identified in the MRC.
  • The MRC was shown to be competent for origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro.
  • Replication requirements, initiation kinetics, and the ability to utilize minichromosome structures were determined.

Conclusions:

  • The human cell MRC is a complex machinery mediating DNA synthesis in mammalian cells.
  • The model for the murine MRC can be extended to include the human cell MRC.
  • Further evidence supports DNA synthesis being mediated by a multiprotein complex in mammalian cells.