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Properties of nucleoprotein complexes containing replicating polyoma DNA
Abstract:
Short-lived nucleoprotein complexes (r-py complex) containing replicating polyoma DNA were isolated from infected cells after lysis with Triton X-100. The Triton lysing procedure of Green, Miller, and Hendler (1971) releases most complexes containing supercoiled viral DNA (py complex) from nuclei, but liberates only a portion of r-py complexes. r-py Complexes are associated more strongly with nuclear sites but can be extracted by prolonged incubation of nuclei in lysing solution. Complexes containing replicating polyoma DNA appear to be precursors to stable complexes containing supercoiled DNA. Sedimentation and buoyant density studies indicate that protein is bound to both r-py complexes and py complexes at a ratio of protein to DNA of about 1 to 2/1. Both types of complexes sediment as if the viral DNA is more compact than free DNA and both undergo major reversible configurational changes with increased salt concentration. Changes resulting from enzymatic and chemical treatment indicate that there may be two or more protein components in both r-py complex and py complex. One component is digested by Pronase and trypsin while another is resistant to the enzymes but released by deoxycholate. The abundance and similarity in chemical and physical properties of protein bound to all forms of polyoma DNA suggest that part of the protein molecules may serve in a structural capacity.
Insights
Researchers isolated short-lived nucleoprotein complexes containing replicating polyoma DNA (r-py complex) and stable supercoiled polyoma DNA (py complex). Protein is bound to both, suggesting a structural role in viral DNA organization within infected cells.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Polyoma virus infection involves the formation of nucleoprotein complexes.
- Understanding these complexes is crucial for deciphering viral DNA replication and nuclear organization.
Purpose of the Study:
- To isolate and characterize short-lived replicating polyoma DNA complexes (r-py complex) and stable supercoiled polyoma DNA complexes (py complex).
- To investigate the protein-DNA interactions and structural properties of these viral nucleoprotein complexes.
Main Methods:
- Isolation of nucleoprotein complexes from infected cells using Triton X-100 lysis.
- Sedimentation and buoyant density studies to analyze complex composition and properties.
- Enzymatic (Pronase, trypsin) and chemical (deoxycholate) treatments to probe protein components.
Main Results:
- Triton lysis releases py complexes but only partially releases r-py complexes, indicating stronger nuclear association of replicating forms.
- r-py complexes appear to be precursors to stable py complexes.
- Both complex types show a protein-to-DNA ratio of approximately 1:2, with DNA appearing more compact than free DNA.
- Complexes exhibit reversible configurational changes with salt concentration.
- Evidence suggests at least two protein components in both complexes, with varying susceptibility to enzymatic and chemical treatments.
Conclusions:
- Protein is bound to both replicating and supercoiled polyoma DNA complexes.
- The properties of bound proteins suggest a significant structural role in organizing viral DNA within the host cell nucleus.
- Replicating polyoma DNA complexes are precursors to stable supercoiled complexes.