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Origin of DNA replication in papovavirus chromatin is recognized by endogenous endonuclease
Abstract:
Isolated simian virus 40 (SV40) and polyoma nucleoprotein complexes contain endonuclease that, under in vitro conditions, converts part (up to 30%) of the covalently closed superhelical DNA to full-length linear rods. The positions of the cleavage sites within the genomes of SV40 and polyoma were determined by digestion with various single-cut restriction endonucleases and subsequent agarose gel electrophoresis of the cleavage products. Both SV40 and polyoma covalently closed superhelical DNA were cleaved open at their respective origins of DNA replication (+/- 75 base pairs). The full-length linear DNA rods whose ends map adjacent to the origin of DNA replication could also be isolated by sodium dodecyl sulfate/phenol extraction both from SV40-infected permissive cells and from purified SV40 virions. These data reveal the presence of a unique structure of the papovavirus chromatin close to the initiation site of DNA replication.
Insights
Simian virus 40 (SV40) and polyoma viruses possess an endonuclease. This enzyme cleaves viral DNA at the replication origin, revealing unique chromatin structures near DNA replication initiation sites.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Papovaviruses, including simian virus 40 (SV40) and polyoma virus, possess unique chromatin structures.
- Understanding the enzymatic activities within these viral nucleoprotein complexes is crucial for elucidating viral DNA replication mechanisms.
Purpose of the Study:
- To identify and characterize the endonuclease activity present in isolated SV40 and polyoma virus nucleoprotein complexes.
- To determine the precise cleavage sites of this endonuclease within the viral genomes.
- To investigate the structural implications of these findings at the origin of DNA replication.
Main Methods:
- Isolation of SV40 and polyoma virus nucleoprotein complexes.
- In vitro DNA cleavage assays using covalently closed superhelical DNA.
- Restriction endonuclease digestion and agarose gel electrophoresis to map cleavage sites.
- Sodium dodecyl sulfate/phenol extraction to isolate linear DNA rods from infected cells and virions.
Main Results:
- Isolated SV40 and polyoma nucleoprotein complexes contain an endonuclease activity.
- This endonuclease converts up to 30% of covalently closed superhelical viral DNA into full-length linear rods.
- Cleavage occurs specifically at the origins of DNA replication for both SV40 and polyoma viruses.
- Linear DNA molecules with ends mapping adjacent to the replication origin were isolated from infected cells and virions.
Conclusions:
- Papovavirus chromatin exhibits a unique structure localized near the origin of DNA replication.
- The identified endonuclease plays a role in processing viral DNA at the replication initiation site.
- These findings provide insights into the structural organization and enzymatic regulation of viral DNA replication.