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Topoisomerase-II-mediated DNA cleavage within the human ribosomal genes
1Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Napoli, Italy.
Abstract:
The interaction of topoisomerase II (topo II) with human ribosomal DNA (rDNA) was investigated in vivo using the antitumoral drug VM26, a specific inhibitor of topo II, that stabilizes the transient cleavable complex. rDNA-protein complexes isolated from nucleoli of TG cells were analyzed for double strand breaks with probes that covered almost all intergenic transcribed spacer (IGS) and all transcribed sequences of tandem repeat ribosomal DNA genes. Preferential cleavage sites were present in only a part of nucleolar rDNA, i.e., the transcribed region. Proteins, purified from the same complexes, were analyzed by Western-blot and stained by an antiserum against both topo II forms, showing the presence of topo II beta.
Insights
Topoisomerase II (topo II) interacts with human ribosomal DNA (rDNA) in cells. This interaction, specifically involving topo II beta, leads to DNA breaks within the transcribed regions of rDNA.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Topoisomerase II (topo II) is crucial for DNA replication and transcription.
- Ribosomal DNA (rDNA) contains genes for ribosomal RNA and is essential for protein synthesis.
- Understanding topo II's role in rDNA is vital for cancer research.
Purpose of the Study:
- To investigate the in vivo interaction between topoisomerase II and human ribosomal DNA.
- To identify specific sites of DNA cleavage mediated by topo II within rDNA.
- To determine the specific form of topo II involved in rDNA interactions.
Main Methods:
- Utilized the topo II inhibitor VM26 to stabilize cleavable complexes in vivo.
- Isolated rDNA-protein complexes from TG cell nucleoli.
- Analyzed DNA double-strand breaks using specific probes for rDNA regions.
- Purified proteins and analyzed them using Western blot with an antiserum against topo II.
Main Results:
- Preferential DNA double-strand breaks were observed in the transcribed regions of nucleolar rDNA.
- The interaction was specific to a portion of the nucleolar rDNA.
- Western blot analysis confirmed the presence of topoisomerase II beta in the rDNA-protein complexes.
Conclusions:
- Topoisomerase II interacts with human ribosomal DNA in vivo.
- The interaction leads to cleavage primarily within the transcribed regions of rDNA.
- Topoisomerase II beta is implicated in the interaction with ribosomal DNA.