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In situ nucleoprotein structure involving origin-proximal SV40 DNA control elements
1Department of Chemistry and Biochemistry, University of California, Los Angeles 90024-1569.
Nucleic Acids Research
|April 11, 1990
Summary
Nucleoprotein structures in SV40 DNA are revealed by nucleases. Permeabilized cells show complex higher-order structures not seen in inactive nuclei, suggesting in vivo organization.
Area of Science:
- Molecular Biology
- Structural Biology
- Virology
Background:
- The SV40 GC-box and adjacent AT-rich regions are crucial for viral DNA replication and transcription.
- Understanding the in vivo nucleoprotein structure is key to deciphering regulatory mechanisms.
- Previous studies indicated functional interactions between T-antigen binding sites and the GC-box.
Purpose of the Study:
- To investigate the in vivo nucleoprotein organization at the SV40 GC-box and AT-rich region.
- To compare nucleoprotein structures in permeabilized cells with those in isolated nuclei.
- To elucidate the role of T-antigen in higher-order nucleoprotein complex formation.
Main Methods:
- Nuclease probing (DNase I) at nucleotide resolution in permeabilized cells and isolated nuclei.
- Analysis of nuclease protection and hypersensitivity patterns.
- Utilizing temperature-sensitive mutants of T-antigen to assess its role.
Main Results:
- Permeabilized cells exhibit distinct nuclease protection and hypersensitivity patterns compared to inactive nuclei.
- Strong nuclease hypersensitive sites in permeabilized cells suggest a higher-order in vivo nucleoprotein complex.
- This higher-order structure is disrupted upon T-antigen inactivation, indicating its critical role.
Conclusions:
- The SV40 GC-box and AT-rich region form a higher-order nucleoprotein complex in vivo.
- This complex organization, involving T-antigen, provides a physical basis for functional interactions of regulatory elements.
- Nuclear preparation methods can alter the native in vivo nucleoprotein structure.