Further characterization of the human cell multiprotein DNA replication complex

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.

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.