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Isolation of 8-methoxypsoralen accessible DNA domains from chromatin of intact cells
1University of Zürich-Tierspital, Institute of Pharmacology and Biochemistry, Switzerland.
Abstract:
The chromatin organization of living mammalian cells was probed using 8-methoxypsoralen (MOP). In intact cells, MOP intercalates into DNA domains which are also preferentially accessible to micrococcal nuclease. After UV365 nm irradiation of MOP-treated cells, this chemical forms bifunctional adducts crosslinking the two strands of DNA. Following extraction of cellular DNA, heat denaturation and renaturation at low temperature, the fraction of crosslinked DNA is obtained following enzymatic hydrolysis of unhybridized, non-crosslinked DNA by nuclease S1 treatment. An application of this procedure in the isolation of 8-methoxypsoralen-accessible DNA domains during DNA excision repair is shown.
Insights
This study uses 8-methoxypsoralen (MOP) to map DNA accessibility in living cells. MOP crosslinking reveals DNA domains involved in cellular processes like DNA repair.
Area of Science:
- Cellular biology
- Molecular genetics
- Biochemistry
Background:
- Understanding chromatin organization is crucial for studying DNA accessibility.
- 8-methoxypsoralen (MOP) is a photoactivated crosslinking agent.
Purpose of the Study:
- To investigate the chromatin organization in living mammalian cells.
- To develop a method for isolating specific DNA domains based on accessibility.
Main Methods:
- Cells were treated with 8-methoxypsoralen (MOP) and irradiated with UV light.
- DNA was extracted, denatured, and crosslinked regions were isolated using S1 nuclease treatment.
- The technique was applied to study DNA domains during excision repair.
Main Results:
- MOP preferentially intercalates into DNA domains accessible to micrococcal nuclease.
- UV irradiation of MOP-treated cells results in DNA strand crosslinking.
- The method successfully isolated MOP-accessible DNA domains during DNA excision repair.
Conclusions:
- 8-methoxypsoralen (MOP) provides a tool to probe DNA accessibility and chromatin organization.
- This technique facilitates the isolation and study of specific DNA domains, including those involved in DNA repair pathways.