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Evidence for binding of extrachromosomal DNA sequences to nuclear matrix proteins in multidrug-resistant KB-V1 cells
F Thiebaut1, A R Hanauske, D D Von Hoff
1University of California San Diego Cancer Center 92103.
Abstract:
Multidrug-resistant KB-V1 cells carry amplified mdrl gene sequences located in an extrachromosomal compartment (on episomes). Since episomes do not contain centromeric or telomeric sequences it is unclear whether they are able to bind to nuclear matrix proteins that may regulate episomal gene expression. Using high salt treatments followed by in situ hybridization and dot blot analyses we found evidence for direct binding of episomal DNA to nuclear matrix proteins. This binding could only be reversed after incubation with trypsin or proteinase K as determined by contour-clamped homogeneous electric field (CHEF) electrophoresis. Our findings are consistent with the concept that circular extrachromosomal DNA may not only reintegrate into nuclear DNA but may also be subject to functional control by regulatory proteins within the nuclear matrix.
Insights
Multidrug-resistant cells contain extrachromosomal DNA (episomes) that bind to nuclear matrix proteins. This binding, reversed by proteinase K, suggests nuclear matrix regulation of episomal gene expression.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Multidrug-resistant KB-V1 cells possess amplified mdr1 gene sequences on episomes.
- The role of nuclear matrix proteins in regulating episomal gene expression is not well understood due to the absence of centromeric and telomeric sequences on episomes.
Purpose of the Study:
- To investigate the binding of episomal DNA to nuclear matrix proteins.
- To determine if nuclear matrix proteins can regulate episomal gene expression.
Main Methods:
- High salt treatments, in situ hybridization, and dot blot analyses were employed to detect episomal DNA binding to nuclear matrix proteins.
- Contour-clamped homogeneous electric field (CHEF) electrophoresis was used to assess the reversibility of this binding after enzymatic treatments.
Main Results:
- Evidence of direct binding between episomal DNA and nuclear matrix proteins was observed.
- This binding was found to be reversible only after incubation with trypsin or proteinase K, indicating a protein-mediated interaction.
Conclusions:
- Circular extrachromosomal DNA (episomes) can directly bind to nuclear matrix proteins.
- This interaction suggests that episomes may be functionally regulated by nuclear matrix proteins, similar to chromosomal DNA.