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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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Localization of PLSCR1 binding to amino acids 1432-1441 of topo IIα. (A) Upper panel, Schematic diagram showing the eight GST-tagged topo IIαCTD fusion proteins used to define the region of topo IIα involved in binding to PLSCR1. Lower panel, NP-40 solubilized HeLa cell lysates were incubated with each of the eight GST-topo IIαCTD fusion proteins and complexes pulled down with GSH-Sepharose beads as described in Materials and Methods section. The proteins were eluted and immunoblotted with PLSCR1 antiserum. (B) Untagged recombinant PLSCR1 was incubated with increasing concentrations of synthetic dodecapeptides corresponding to topo IIα residues 1430-1441 or as a control, randomly scrambled topo IIα residues 1430-1441, and GST-topo IIα1258-1531 or GST alone (control). Complexes were pulled down with GSH-Sepharose beads and bound proteins analysed by immunoblotting with PLSCR1 antiserum. The experiment shown was repeated three or more times with comparable results. (C) The sequences of the COOH-termini of topo IIα and topo IIβ were aligned using ClustalW. The basic conserved putative PLSCR1 binding motifs in topo IIα and topo IIβ are boxed; the vertical bars in the topo II sequences represent intron/exon boundaries (43), and the functional NLS sequences in this region are underlined (topo IIα, solid line; topo IIβ, broken line) (24).
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Figure 6: Localization of PLSCR1 binding to amino acids 1432-1441 of topo IIα. (A) Upper panel, Schematic diagram showing the eight GST-tagged topo IIαCTD fusion proteins used to define the region of topo IIα involved in binding to PLSCR1. Lower panel, NP-40 solubilized HeLa cell lysates were incubated with each of the eight GST-topo IIαCTD fusion proteins and complexes pulled down with GSH-Sepharose beads as described in Materials and Methods section. The proteins were eluted and immunoblotted with PLSCR1 antiserum. (B) Untagged recombinant PLSCR1 was incubated with increasing concentrations of synthetic dodecapeptides corresponding to topo IIα residues 1430-1441 or as a control, randomly scrambled topo IIα residues 1430-1441, and GST-topo IIα1258-1531 or GST alone (control). Complexes were pulled down with GSH-Sepharose beads and bound proteins analysed by immunoblotting with PLSCR1 antiserum. The experiment shown was repeated three or more times with comparable results. (C) The sequences of the COOH-termini of topo IIα and topo IIβ were aligned using ClustalW. The basic conserved putative PLSCR1 binding motifs in topo IIα and topo IIβ are boxed; the vertical bars in the topo II sequences represent intron/exon boundaries (43), and the functional NLS sequences in this region are underlined (topo IIα, solid line; topo IIβ, broken line) (24).

Mentions: To identify the region of topo IIα that mediates binding to PLSCR1, eight GST fusion proteins encoding NH2– and COOH– truncated forms of the topo IIα CTD (Figure 6A, upper panel) were generated and tested for their ability to bind endogenous PLSCR1 in a GST-pull-down assay. Only one of these fusion proteins (topo IIα1171-1431) failed to bind to endogenous PLSCR1, indicating that topo IIα residues 1432-1441 are required for binding to PLSCR1 (Figure 6A, lower panel).Figure 6.


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

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

Localization of PLSCR1 binding to amino acids 1432-1441 of topo IIα. (A) Upper panel, Schematic diagram showing the eight GST-tagged topo IIαCTD fusion proteins used to define the region of topo IIα involved in binding to PLSCR1. Lower panel, NP-40 solubilized HeLa cell lysates were incubated with each of the eight GST-topo IIαCTD fusion proteins and complexes pulled down with GSH-Sepharose beads as described in Materials and Methods section. The proteins were eluted and immunoblotted with PLSCR1 antiserum. (B) Untagged recombinant PLSCR1 was incubated with increasing concentrations of synthetic dodecapeptides corresponding to topo IIα residues 1430-1441 or as a control, randomly scrambled topo IIα residues 1430-1441, and GST-topo IIα1258-1531 or GST alone (control). Complexes were pulled down with GSH-Sepharose beads and bound proteins analysed by immunoblotting with PLSCR1 antiserum. The experiment shown was repeated three or more times with comparable results. (C) The sequences of the COOH-termini of topo IIα and topo IIβ were aligned using ClustalW. The basic conserved putative PLSCR1 binding motifs in topo IIα and topo IIβ are boxed; the vertical bars in the topo II sequences represent intron/exon boundaries (43), and the functional NLS sequences in this region are underlined (topo IIα, solid line; topo IIβ, broken line) (24).
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Figure 6: Localization of PLSCR1 binding to amino acids 1432-1441 of topo IIα. (A) Upper panel, Schematic diagram showing the eight GST-tagged topo IIαCTD fusion proteins used to define the region of topo IIα involved in binding to PLSCR1. Lower panel, NP-40 solubilized HeLa cell lysates were incubated with each of the eight GST-topo IIαCTD fusion proteins and complexes pulled down with GSH-Sepharose beads as described in Materials and Methods section. The proteins were eluted and immunoblotted with PLSCR1 antiserum. (B) Untagged recombinant PLSCR1 was incubated with increasing concentrations of synthetic dodecapeptides corresponding to topo IIα residues 1430-1441 or as a control, randomly scrambled topo IIα residues 1430-1441, and GST-topo IIα1258-1531 or GST alone (control). Complexes were pulled down with GSH-Sepharose beads and bound proteins analysed by immunoblotting with PLSCR1 antiserum. The experiment shown was repeated three or more times with comparable results. (C) The sequences of the COOH-termini of topo IIα and topo IIβ were aligned using ClustalW. The basic conserved putative PLSCR1 binding motifs in topo IIα and topo IIβ are boxed; the vertical bars in the topo II sequences represent intron/exon boundaries (43), and the functional NLS sequences in this region are underlined (topo IIα, solid line; topo IIβ, broken line) (24).
Mentions: To identify the region of topo IIα that mediates binding to PLSCR1, eight GST fusion proteins encoding NH2– and COOH– truncated forms of the topo IIα CTD (Figure 6A, upper panel) were generated and tested for their ability to bind endogenous PLSCR1 in a GST-pull-down assay. Only one of these fusion proteins (topo IIα1171-1431) failed to bind to endogenous PLSCR1, indicating that topo IIα residues 1432-1441 are required for binding to PLSCR1 (Figure 6A, lower panel).Figure 6.

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