Related Experiment Videos
Decreased expression of topoisomerase IIbeta in poly(ADP-ribose) polymerase-deficient cells
Y Canitrot1, G de Murcia, B Salles
1Institut de Pharmacologie et Biologie Structurale, UPR CNRS 9062, 205 route de Narbonne, 31077 Toulouse cedex, France.
Abstract:
Recent studies with poly(ADP-ribose) polymerase (PARP)-deficient mice have highlighted the role of this enzyme in genomic stability and response to various genomic insults. In the absence of DNA damaging treatment, we report here that a PARP-deficient cell line (PARP-/-) established from knockout mice displays a decrease in topoisomerase II (topo II) activity as measured by decatenation of kinetoplast DNA. Immunoblotting of whole and nuclear cell extracts showed that reduced activity was associated with decreased amount of the 180 kDa topo IIbeta protein but not of the 170 kDa topo IIalpha. The decreased topo IIbeta expression did not stem from transcriptional regulation of gene expression since levels of topo IIbeta mRNA were similar in PARP (-/-) compared with the parental PARP (+/+) cells. The decreased topo II activity was associated with cell resistance to VP16, a topo II inhibitor. These observations indicate that PARP may play a role in the stabilization and/or distribution of topo IIbeta.
Insights
Poly(ADP-ribose) polymerase (PARP) deficiency reduces topoisomerase II (topo II) activity by decreasing topo IIbeta protein levels, independent of gene expression. This impacts genomic stability and drug response.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- Poly(ADP-ribose) polymerase (PARP) is crucial for genomic stability and DNA damage response.
- PARP-deficient mice models have illuminated its role in cellular repair mechanisms.
Purpose of the Study:
- To investigate the intrinsic activity of topoisomerase II (topo II) in PARP-deficient cells.
- To determine the molecular basis for any observed changes in topo II activity.
Main Methods:
- Utilized a PARP-deficient cell line (PARP-/-) derived from knockout mice.
- Assessed topo II activity via kinetoplast DNA decatenation assays.
- Quantified topo IIalpha and topo IIbeta protein levels using immunoblotting.
- Measured topo IIbeta mRNA levels to assess transcriptional regulation.
Main Results:
- PARP-deficient cells exhibited reduced topo II activity without DNA damage.
- This reduction correlated with decreased levels of the 180 kDa topo IIbeta protein, not topo IIalpha.
- Topo IIbeta mRNA levels remained unchanged, indicating post-transcriptional regulation.
- PARP deficiency led to increased resistance to the topo II inhibitor VP16.
Conclusions:
- PARP plays a significant role in maintaining topoisomerase IIbeta protein stability and/or distribution.
- The absence of PARP affects basal topo II activity, influencing genomic integrity.
- These findings suggest a novel regulatory link between PARP and topo II function.