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Requirement of DNA topoisomerases for in vitro chromatin assembly by 3T6 mouse cell extracts
J F Cáceres1, D Blangy, G C Glikin
1Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Universidad de Buenos Aires, Argentina.
Abstract:
Nuclear extracts of 3T6 mouse cells were able to assemble in vitro minichromosomes which displayed a 150-bp periodicity. Activities of both DNA topoisomerases I and II were detected in these extracts. When a supercoiled pUC DNA was added, it was first relaxed in less than 3 min, then slowly supercoiled again in 1-4 h. Both reactions occurred either in the absence or the presence of added Mg2+ and/or ATP, they were not blocked by DNA topoisomerase II inhibitors and they were inhibited by an antiserum against DNA topoisomerase I and by camptothecin. These findings led us to propose that, under our in vitro assay conditions, chromatin assembly is mainly carried out by a DNA topoisomerase I.
Insights
This study shows that DNA topoisomerase I, not II, is the primary enzyme responsible for chromatin assembly in vitro. This finding clarifies the enzymatic mechanisms driving DNA organization in cellular processes.
Area of Science:
- Molecular Biology
- Biochemistry
- Epigenetics
Background:
- Chromatin assembly is crucial for DNA packaging and regulation.
- Nuclear extracts contain enzymes that manage DNA topology.
- The roles of DNA topoisomerases in chromatin assembly are not fully elucidated.
Purpose of the Study:
- To investigate the enzymatic activity responsible for in vitro chromatin assembly.
- To determine the specific roles of DNA topoisomerases I and II in this process.
Main Methods:
- Utilized nuclear extracts from 3T6 mouse cells for in vitro assays.
- Introduced supercoiled pUC DNA to monitor topological changes.
- Assessed the effects of enzyme inhibitors and specific antisera.
Main Results:
- Nuclear extracts assembled minichromosomes with 150-bp periodicity.
- DNA topoisomerases I and II activities were detected.
- Supercoiled DNA was relaxed and then slowly supercoiled.
- These reactions were insensitive to topoisomerase II inhibitors but sensitive to topoisomerase I inhibition.
Conclusions:
- DNA topoisomerase I is the predominant enzyme driving in vitro chromatin assembly.
- This highlights the specific role of topoisomerase I in DNA organization and packaging.