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DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK.

Bigot S, Marians KJ - Nucleic Acids Res. (2010)

Bottom Line: DNA chirality did not affect any of the activities of FtsK, suggesting that FtsK possesses an inherent Topo IV stimulatory activity that is presumably mediated by protein-protein interactions, the stability of Topo IV on the DNA substrate dictated the effect observed.Inhibition occurs because FtsK can strip distributively acting topoisomerase off (-)ve scDNA, but not from either (+)ve scDNA or catenated DNA where the enzyme acts processively.Our analyses suggest that FtsK increases the efficiency of trapping of the transfer segment of DNA during the catalytic cycle of the topoisomerase.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

ABSTRACT
We have studied the stimulation of topoisomerase IV (Topo IV) by the C-terminal AAA+ domain of FtsK. These two proteins combine to assure proper chromosome segregation in the cell. Stimulation of Topo IV activity was dependent on the chirality of the DNA substrate: FtsK stimulated decatenation of catenated DNA and relaxation of positively supercoiled [(+)ve sc] DNA, but inhibited relaxation of negatively supercoiled [(-)ve sc] DNA. The DNA translocation activity of FtsK was not required for stimulation, but was required for inhibition. DNA chirality did not affect any of the activities of FtsK, suggesting that FtsK possesses an inherent Topo IV stimulatory activity that is presumably mediated by protein-protein interactions, the stability of Topo IV on the DNA substrate dictated the effect observed. Inhibition occurs because FtsK can strip distributively acting topoisomerase off (-)ve scDNA, but not from either (+)ve scDNA or catenated DNA where the enzyme acts processively. Our analyses suggest that FtsK increases the efficiency of trapping of the transfer segment of DNA during the catalytic cycle of the topoisomerase.

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Effect of FtsK50C on the catalytic mechanism of Topo IV. (A) Reaction mixtures containing either no NTP or 2 mM AMP-PNP, 7.5 nM Topo IV and 5 nM (+)ve scDNA were incubated and analyzed as described under ‘Experimental Procedures’ section. The fraction of DNA cleaved is presented (average of three independent experiments). (B) FtsK does not alter the DNA cleavage–religation equilibrium of Topo IV. Reaction mixtures containing 2 mM AMP-PNP, 4 nM Topo IV, 5 nM (+)ve scDNA and 60 nM of either FtsK50C or FtsK50CK997A were incubated and analyzed as described under ‘Experimental Procedures’ section. The fraction of DNA cleaved is presented (the average of three independent experiments). (C) FtsK50CK997A does not affect the ATPase activity of Topo IV. Reaction mixtures containing the indicated amounts of Topo IV, 5 nM (+)ve sc DNA, [α-32P]ATP and either no FtsK50CK997A or 60 nM FtsK50CK997A were incubated and analyzed as described under ‘Experimental Procedures’ section. The results of three independent experiments were averaged.
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Figure 3: Effect of FtsK50C on the catalytic mechanism of Topo IV. (A) Reaction mixtures containing either no NTP or 2 mM AMP-PNP, 7.5 nM Topo IV and 5 nM (+)ve scDNA were incubated and analyzed as described under ‘Experimental Procedures’ section. The fraction of DNA cleaved is presented (average of three independent experiments). (B) FtsK does not alter the DNA cleavage–religation equilibrium of Topo IV. Reaction mixtures containing 2 mM AMP-PNP, 4 nM Topo IV, 5 nM (+)ve scDNA and 60 nM of either FtsK50C or FtsK50CK997A were incubated and analyzed as described under ‘Experimental Procedures’ section. The fraction of DNA cleaved is presented (the average of three independent experiments). (C) FtsK50CK997A does not affect the ATPase activity of Topo IV. Reaction mixtures containing the indicated amounts of Topo IV, 5 nM (+)ve sc DNA, [α-32P]ATP and either no FtsK50CK997A or 60 nM FtsK50CK997A were incubated and analyzed as described under ‘Experimental Procedures’ section. The results of three independent experiments were averaged.

Mentions: To determine whether the presence of FtsK50C affected the Topo IV cleavage–religation equilibrium, we analyzed the effect of FtsK50C on the ability of the Topo IV to bind DNA and make a double-strand DNA break. Topo IV was incubated with DNA and AMP-PNP in either the presence or absence of FtsK50C, SDS was added to denature cleavable complexes, the DNA products were treated with proteinase K and then analyzed by gel electrophoresis (Figure 3B). Neither FtsK50C nor FtsK50CK997A affected Topo IV-mediated DNA cleavage.Figure 3.


DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK.

Bigot S, Marians KJ - Nucleic Acids Res. (2010)

Effect of FtsK50C on the catalytic mechanism of Topo IV. (A) Reaction mixtures containing either no NTP or 2 mM AMP-PNP, 7.5 nM Topo IV and 5 nM (+)ve scDNA were incubated and analyzed as described under ‘Experimental Procedures’ section. The fraction of DNA cleaved is presented (average of three independent experiments). (B) FtsK does not alter the DNA cleavage–religation equilibrium of Topo IV. Reaction mixtures containing 2 mM AMP-PNP, 4 nM Topo IV, 5 nM (+)ve scDNA and 60 nM of either FtsK50C or FtsK50CK997A were incubated and analyzed as described under ‘Experimental Procedures’ section. The fraction of DNA cleaved is presented (the average of three independent experiments). (C) FtsK50CK997A does not affect the ATPase activity of Topo IV. Reaction mixtures containing the indicated amounts of Topo IV, 5 nM (+)ve sc DNA, [α-32P]ATP and either no FtsK50CK997A or 60 nM FtsK50CK997A were incubated and analyzed as described under ‘Experimental Procedures’ section. The results of three independent experiments were averaged.
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Figure 3: Effect of FtsK50C on the catalytic mechanism of Topo IV. (A) Reaction mixtures containing either no NTP or 2 mM AMP-PNP, 7.5 nM Topo IV and 5 nM (+)ve scDNA were incubated and analyzed as described under ‘Experimental Procedures’ section. The fraction of DNA cleaved is presented (average of three independent experiments). (B) FtsK does not alter the DNA cleavage–religation equilibrium of Topo IV. Reaction mixtures containing 2 mM AMP-PNP, 4 nM Topo IV, 5 nM (+)ve scDNA and 60 nM of either FtsK50C or FtsK50CK997A were incubated and analyzed as described under ‘Experimental Procedures’ section. The fraction of DNA cleaved is presented (the average of three independent experiments). (C) FtsK50CK997A does not affect the ATPase activity of Topo IV. Reaction mixtures containing the indicated amounts of Topo IV, 5 nM (+)ve sc DNA, [α-32P]ATP and either no FtsK50CK997A or 60 nM FtsK50CK997A were incubated and analyzed as described under ‘Experimental Procedures’ section. The results of three independent experiments were averaged.
Mentions: To determine whether the presence of FtsK50C affected the Topo IV cleavage–religation equilibrium, we analyzed the effect of FtsK50C on the ability of the Topo IV to bind DNA and make a double-strand DNA break. Topo IV was incubated with DNA and AMP-PNP in either the presence or absence of FtsK50C, SDS was added to denature cleavable complexes, the DNA products were treated with proteinase K and then analyzed by gel electrophoresis (Figure 3B). Neither FtsK50C nor FtsK50CK997A affected Topo IV-mediated DNA cleavage.Figure 3.

Bottom Line: DNA chirality did not affect any of the activities of FtsK, suggesting that FtsK possesses an inherent Topo IV stimulatory activity that is presumably mediated by protein-protein interactions, the stability of Topo IV on the DNA substrate dictated the effect observed.Inhibition occurs because FtsK can strip distributively acting topoisomerase off (-)ve scDNA, but not from either (+)ve scDNA or catenated DNA where the enzyme acts processively.Our analyses suggest that FtsK increases the efficiency of trapping of the transfer segment of DNA during the catalytic cycle of the topoisomerase.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

ABSTRACT
We have studied the stimulation of topoisomerase IV (Topo IV) by the C-terminal AAA+ domain of FtsK. These two proteins combine to assure proper chromosome segregation in the cell. Stimulation of Topo IV activity was dependent on the chirality of the DNA substrate: FtsK stimulated decatenation of catenated DNA and relaxation of positively supercoiled [(+)ve sc] DNA, but inhibited relaxation of negatively supercoiled [(-)ve sc] DNA. The DNA translocation activity of FtsK was not required for stimulation, but was required for inhibition. DNA chirality did not affect any of the activities of FtsK, suggesting that FtsK possesses an inherent Topo IV stimulatory activity that is presumably mediated by protein-protein interactions, the stability of Topo IV on the DNA substrate dictated the effect observed. Inhibition occurs because FtsK can strip distributively acting topoisomerase off (-)ve scDNA, but not from either (+)ve scDNA or catenated DNA where the enzyme acts processively. Our analyses suggest that FtsK increases the efficiency of trapping of the transfer segment of DNA during the catalytic cycle of the topoisomerase.

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