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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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PLSCR1 stimulates the decatenation activity of topo IIα. Topo II decatenating activity was assayed using purified topo IIα enzyme in the presence and absence of untagged recombinant PLSCR1 as described in Materials and Methods section. Similar results to those shown were obtained in two additional experiments. The positions of the catenated kDNA (cat kDNA), decatenated kDNA minicircles (dec) and linear (lin) kDNA are indicated.
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Figure 5: PLSCR1 stimulates the decatenation activity of topo IIα. Topo II decatenating activity was assayed using purified topo IIα enzyme in the presence and absence of untagged recombinant PLSCR1 as described in Materials and Methods section. Similar results to those shown were obtained in two additional experiments. The positions of the catenated kDNA (cat kDNA), decatenated kDNA minicircles (dec) and linear (lin) kDNA are indicated.

Mentions: The ability of PLSCR1 to modulate topo II activity was assessed using a topo II assay that measures the decatenating activity of the enzyme. Thus purified topo IIα was preincubated with PLSCR1 for 1.5 h at 20°C prior to incubation with kDNA for 15 min at 37°C. As shown in Figure 5, the catenated kDNA did not enter the gel (lane 5) as expected, while decatenated DNA entered the gel, but its electrophoretic mobility was slightly retarded compared to linear DNA (compare lanes 6 and 7). The addition of recombinant PLSCR1 to the assay mixture significantly increased the ability of topo IIα to decatenate kDNA (compare lanes 3 and 4 versus 1 and 2).Figure 5.


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

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

PLSCR1 stimulates the decatenation activity of topo IIα. Topo II decatenating activity was assayed using purified topo IIα enzyme in the presence and absence of untagged recombinant PLSCR1 as described in Materials and Methods section. Similar results to those shown were obtained in two additional experiments. The positions of the catenated kDNA (cat kDNA), decatenated kDNA minicircles (dec) and linear (lin) kDNA are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: PLSCR1 stimulates the decatenation activity of topo IIα. Topo II decatenating activity was assayed using purified topo IIα enzyme in the presence and absence of untagged recombinant PLSCR1 as described in Materials and Methods section. Similar results to those shown were obtained in two additional experiments. The positions of the catenated kDNA (cat kDNA), decatenated kDNA minicircles (dec) and linear (lin) kDNA are indicated.
Mentions: The ability of PLSCR1 to modulate topo II activity was assessed using a topo II assay that measures the decatenating activity of the enzyme. Thus purified topo IIα was preincubated with PLSCR1 for 1.5 h at 20°C prior to incubation with kDNA for 15 min at 37°C. As shown in Figure 5, the catenated kDNA did not enter the gel (lane 5) as expected, while decatenated DNA entered the gel, but its electrophoretic mobility was slightly retarded compared to linear DNA (compare lanes 6 and 7). The addition of recombinant PLSCR1 to the assay mixture significantly increased the ability of topo IIα to decatenate kDNA (compare lanes 3 and 4 versus 1 and 2).Figure 5.

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