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Nuclear interactions of topoisomerase II alpha and beta with phospholipid scramblase 1.

Wyles JP, Wu Z, Mirski SE, Cole SP - Nucleic Acids Res. (2007)

Bottom Line: PLSCR1 also increased the decatenation activity of human topo IIalpha.A conserved basic sequence in the CTD of topo IIalpha was identified as being essential for binding to PLSCR1 and binding of the two proteins could be inhibited by a synthetic peptide corresponding to topo IIalpha amino acids 1430-1441.These studies reveal for the first time a physical and functional interaction between topo II and PLSCR1.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, Ontario, Canada K7L 3N6.

ABSTRACT
DNA topoisomerase (topo) II modulates DNA topology and is essential for cell division. There are two isoforms of topo II (alpha and beta) that have limited functional redundancy, although their catalytic mechanisms appear the same. Using their COOH-terminal domains (CTDs) in yeast two-hybrid analysis, we have identified phospholipid scramblase 1 (PLSCR1) as a binding partner of both topo II alpha and beta. Although predominantly a plasma membrane protein involved in phosphatidylserine externalization, PLSCR1 can also be imported into the nucleus where it may have a tumour suppressor function. The interactions of PLSCR1 and topo II were confirmed by pull-down assays with topo II alpha and beta CTD fusion proteins and endogenous PLSCR1, and by co-immunoprecipitation of endogenous PLSCR1 and topo II alpha and beta from HeLa cell nuclear extracts. PLSCR1 also increased the decatenation activity of human topo IIalpha. A conserved basic sequence in the CTD of topo IIalpha was identified as being essential for binding to PLSCR1 and binding of the two proteins could be inhibited by a synthetic peptide corresponding to topo IIalpha amino acids 1430-1441. These studies reveal for the first time a physical and functional interaction between topo II and PLSCR1.

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Endogenous topo II α and β co-immunoprecipitate with endogenous PLSCR1. Nuclear extracts prepared from HeLa cells were subjected to co-immunoprecipitation with an anti-PLSCR1 antiserum or pre-immune serum as described in Materials and Methods section. Immunoprecipitated samples were then analysed by immunoblotting with topo II α and β specific mAbs 8D2 and 3H10, respectively. Input lanes represent 5% of sample used in the assay. Shown are results from a representative experiment and similar results were obtained in two additional experiments.
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Figure 3: Endogenous topo II α and β co-immunoprecipitate with endogenous PLSCR1. Nuclear extracts prepared from HeLa cells were subjected to co-immunoprecipitation with an anti-PLSCR1 antiserum or pre-immune serum as described in Materials and Methods section. Immunoprecipitated samples were then analysed by immunoblotting with topo II α and β specific mAbs 8D2 and 3H10, respectively. Input lanes represent 5% of sample used in the assay. Shown are results from a representative experiment and similar results were obtained in two additional experiments.

Mentions: To confirm that endogenous topo II α and β interact with endogenous PLSCR1 in intact cells, nuclear lysates were subjected to immunoprecipitation analysis using a PLSCR1 antiserum, followed by immunoblotting with topo II α and β specific mAbs. As shown in Figure 3, both topo II α and β were immunoprecipitated by the PLSCR1 antiserum, but not the pre-immune serum, demonstrating that the endogenous proteins interact in vivo. These results also show that the interaction is not an artifact caused by the use of recombinant topo II CTDs rather than the endogenous proteins, or the forced colocalization of proteins that do not normally share a compartment, as can occur in yeast two-hybrid analysis. The input lane represents 5% of the nuclear lysate used in each binding assay, suggesting that as much as 5% of nuclear topo II α and β is associated with PLSCR1. A high background associated with the PLSCR1 antiserum in co-immunoprecipitation experiments precluded consistent western blot detection of nuclear PLSCR1 immunoprecipitated by the topo II α and β specific mAbs (data not shown).Figure 3.


Nuclear interactions of topoisomerase II alpha and beta with phospholipid scramblase 1.

Wyles JP, Wu Z, Mirski SE, Cole SP - Nucleic Acids Res. (2007)

Endogenous topo II α and β co-immunoprecipitate with endogenous PLSCR1. Nuclear extracts prepared from HeLa cells were subjected to co-immunoprecipitation with an anti-PLSCR1 antiserum or pre-immune serum as described in Materials and Methods section. Immunoprecipitated samples were then analysed by immunoblotting with topo II α and β specific mAbs 8D2 and 3H10, respectively. Input lanes represent 5% of sample used in the assay. Shown are results from a representative experiment and similar results were obtained in two additional experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919507&req=5

Figure 3: Endogenous topo II α and β co-immunoprecipitate with endogenous PLSCR1. Nuclear extracts prepared from HeLa cells were subjected to co-immunoprecipitation with an anti-PLSCR1 antiserum or pre-immune serum as described in Materials and Methods section. Immunoprecipitated samples were then analysed by immunoblotting with topo II α and β specific mAbs 8D2 and 3H10, respectively. Input lanes represent 5% of sample used in the assay. Shown are results from a representative experiment and similar results were obtained in two additional experiments.
Mentions: To confirm that endogenous topo II α and β interact with endogenous PLSCR1 in intact cells, nuclear lysates were subjected to immunoprecipitation analysis using a PLSCR1 antiserum, followed by immunoblotting with topo II α and β specific mAbs. As shown in Figure 3, both topo II α and β were immunoprecipitated by the PLSCR1 antiserum, but not the pre-immune serum, demonstrating that the endogenous proteins interact in vivo. These results also show that the interaction is not an artifact caused by the use of recombinant topo II CTDs rather than the endogenous proteins, or the forced colocalization of proteins that do not normally share a compartment, as can occur in yeast two-hybrid analysis. The input lane represents 5% of the nuclear lysate used in each binding assay, suggesting that as much as 5% of nuclear topo II α and β is associated with PLSCR1. A high background associated with the PLSCR1 antiserum in co-immunoprecipitation experiments precluded consistent western blot detection of nuclear PLSCR1 immunoprecipitated by the topo II α and β specific mAbs (data not shown).Figure 3.

Bottom Line: PLSCR1 also increased the decatenation activity of human topo IIalpha.A conserved basic sequence in the CTD of topo IIalpha was identified as being essential for binding to PLSCR1 and binding of the two proteins could be inhibited by a synthetic peptide corresponding to topo IIalpha amino acids 1430-1441.These studies reveal for the first time a physical and functional interaction between topo II and PLSCR1.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, Ontario, Canada K7L 3N6.

ABSTRACT
DNA topoisomerase (topo) II modulates DNA topology and is essential for cell division. There are two isoforms of topo II (alpha and beta) that have limited functional redundancy, although their catalytic mechanisms appear the same. Using their COOH-terminal domains (CTDs) in yeast two-hybrid analysis, we have identified phospholipid scramblase 1 (PLSCR1) as a binding partner of both topo II alpha and beta. Although predominantly a plasma membrane protein involved in phosphatidylserine externalization, PLSCR1 can also be imported into the nucleus where it may have a tumour suppressor function. The interactions of PLSCR1 and topo II were confirmed by pull-down assays with topo II alpha and beta CTD fusion proteins and endogenous PLSCR1, and by co-immunoprecipitation of endogenous PLSCR1 and topo II alpha and beta from HeLa cell nuclear extracts. PLSCR1 also increased the decatenation activity of human topo IIalpha. A conserved basic sequence in the CTD of topo IIalpha was identified as being essential for binding to PLSCR1 and binding of the two proteins could be inhibited by a synthetic peptide corresponding to topo IIalpha amino acids 1430-1441. These studies reveal for the first time a physical and functional interaction between topo II and PLSCR1.

Show MeSH
Related in: MedlinePlus