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A 17S multiprotein form of murine cell DNA polymerase mediates polyomavirus DNA replication in vitro
1Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore 21201.
Abstract:
We have identified and purified a multiprotein form of DNA polymerase from the murine mammary carcinoma cell line (FM3A) using a series of centrifugation, polyethylene glycol precipitation, and ion-exchange chromatography steps. Proteins and enzymatic activities associated with this mouse cell multiprotein form of DNA polymerase include the DNA polymerases alpha and delta, DNA primase, proliferating cell nuclear antigen (PCNA), DNA ligase I, DNA helicase, and DNA topoisomerases I and II. The sedimentation coefficient of the multiprotein form of DNA polymerase is 17S, as determined by sucrose density gradient analysis. The integrity of the murine cell multiprotein form of DNA polymerase is maintained after treatment with detergents, salt, RNase, DNase, and after chromatography on DE52-cellulose, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecular components. Most importantly, we have demonstrated that this complex of proteins is fully competent to replicate polyomavirus DNA in vitro. This result implies that all of the cellular activities required for large T-antigen dependent in vitro polyomavirus DNA synthesis are present within the isolated 17S multiprotein form of the mouse cell DNA replication activities. A model is proposed to represent the mammalian Multiprotein DNA Replication Complex (MRC) based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex.
Insights
Researchers purified a multiprotein DNA replication complex from mouse cells, containing key enzymes like DNA polymerase alpha and delta. This complex efficiently replicates polyomavirus DNA in vitro, suggesting it contains all necessary factors for mammalian DNA synthesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- DNA replication is a fundamental cellular process essential for cell division and organismal development.
- Mammalian DNA replication involves a complex interplay of numerous proteins and enzymes.
- Understanding the composition and function of DNA replication machinery is crucial for comprehending cell cycle regulation and diseases like cancer.
Purpose of the Study:
- To identify and purify the multiprotein DNA polymerase complex from murine mammary carcinoma cells (FM3A).
- To characterize the protein and enzymatic components of the purified complex.
- To assess the in vitro DNA replication capability of the isolated complex and propose a model for the mammalian replication machinery.
Main Methods:
- Purification using centrifugation, polyethylene glycol precipitation, and ion-exchange chromatography.
- Sucrose density gradient analysis to determine sedimentation coefficient (17S).
- Enzymatic assays and protein identification to characterize complex components.
Main Results:
- A 17S multiprotein complex containing DNA polymerases alpha and delta, DNA primase, PCNA, DNA ligase I, helicase, and topoisomerases I and II was isolated.
- The complex's integrity was stable after various treatments (detergents, salt, nucleases, chromatography).
- The purified complex demonstrated full competence in replicating polyomavirus DNA in vitro.
Conclusions:
- The isolated 17S complex represents a functional mammalian DNA replication complex (MRC).
- This MRC contains all essential cellular activities for large T-antigen dependent in vitro polyomavirus DNA synthesis.
- A model for the mammalian MRC is proposed based on the co-purified protein components and their activities.