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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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FtsK50C, but not FtsK50CK997A, inhibits the DNA relaxation activity of ParC(ΔCTD) Topo IV. Reaction mixtures containing the indicated amounts of ParC(ΔCTD)Topo IV, either FtsK50C or FtsK50CK997A, and either (+)ve scDNA (A and C, respectively) or (−)ve sc DNA (B and D, respectively) were incubated and analyzed as described under ‘Experimental Procedures’ section. ParC(ΔCTD) Topo IV, Topo IV reconstituted with wild-type ParE and ParC(ΔCTD).
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Figure 6: FtsK50C, but not FtsK50CK997A, inhibits the DNA relaxation activity of ParC(ΔCTD) Topo IV. Reaction mixtures containing the indicated amounts of ParC(ΔCTD)Topo IV, either FtsK50C or FtsK50CK997A, and either (+)ve scDNA (A and C, respectively) or (−)ve sc DNA (B and D, respectively) were incubated and analyzed as described under ‘Experimental Procedures’ section. ParC(ΔCTD) Topo IV, Topo IV reconstituted with wild-type ParE and ParC(ΔCTD).

Mentions: Much of the discrimination shown by Topo IV between DNA substrates can be attributed to the CTD of ParC. Removal of this region of ParC results in a slight overall reduction of the affinity of the variant Topo IV for all DNA substrates (binding to the G segment) and a dramatic loss of topological discrimination (binding to the T segment) (11). CTD-truncated (residues 1–482) ParC was purified. The reconstituted ParC(ΔCTD) Topo IV exhibited reduced activity compared with wild-type Topo IV and lost discrimination between DNAs of opposite chirality, as shown previously by Corbett et al. (11). Given that the activity of ParC(ΔCTD) Topo IV is independent of the substrate, we predicted that the FtsK50C derivatives would have equivalent effects on the relaxation of (+)ve and (−)ve sc DNA by Topo IV. This proved to be the case. FtsK50C now inhibited, rather than stimulated, ParC(ΔCTD) Topo IV relaxation of (+)ve scDNA (Figure 6A) while retaining its ability to inhibit relaxation of (−)ve sc DNA (Figure 6B). FtsK50CK997A no longer stimulated relaxation of (+)ve scDNA, but also had no effect on either the (+)ve or (−)ve sc DNA relaxation activity of ParC(ΔCTD) Topo IV (Figure 6C and D, respectively). The increased concentrations of ParC(ΔCTD) Topo IV required to establish relaxation of the DNA substrates suggest a roughly 80- to 120-fold decrease in activity. This observation is consonant with the demonstration by Stone et al. (33) that relaxation of positive supercoils by Topo IV has an inherent processivity of ∼80 strand passage events, as well as with the observation of Neuman et al. (33) that relaxation of negative supercoils is perfectly distributive. Because ParC(ΔCTD) Topo IV can no longer discriminate topology, its activity is expected to be distributive [this is apparent in the data of Corbett et al. (11)]. We suggest that because of this change in activity, ParC(ΔCTD) Topo IV can no longer establish a stimulatory complex with either FtsK50C or FtsK50CK997A and that this destabilized ParC(ΔCTD) Topo IV can be displaced by translocating wild-type FtsK50C.Figure 6.


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

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

FtsK50C, but not FtsK50CK997A, inhibits the DNA relaxation activity of ParC(ΔCTD) Topo IV. Reaction mixtures containing the indicated amounts of ParC(ΔCTD)Topo IV, either FtsK50C or FtsK50CK997A, and either (+)ve scDNA (A and C, respectively) or (−)ve sc DNA (B and D, respectively) were incubated and analyzed as described under ‘Experimental Procedures’ section. ParC(ΔCTD) Topo IV, Topo IV reconstituted with wild-type ParE and ParC(ΔCTD).
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Figure 6: FtsK50C, but not FtsK50CK997A, inhibits the DNA relaxation activity of ParC(ΔCTD) Topo IV. Reaction mixtures containing the indicated amounts of ParC(ΔCTD)Topo IV, either FtsK50C or FtsK50CK997A, and either (+)ve scDNA (A and C, respectively) or (−)ve sc DNA (B and D, respectively) were incubated and analyzed as described under ‘Experimental Procedures’ section. ParC(ΔCTD) Topo IV, Topo IV reconstituted with wild-type ParE and ParC(ΔCTD).
Mentions: Much of the discrimination shown by Topo IV between DNA substrates can be attributed to the CTD of ParC. Removal of this region of ParC results in a slight overall reduction of the affinity of the variant Topo IV for all DNA substrates (binding to the G segment) and a dramatic loss of topological discrimination (binding to the T segment) (11). CTD-truncated (residues 1–482) ParC was purified. The reconstituted ParC(ΔCTD) Topo IV exhibited reduced activity compared with wild-type Topo IV and lost discrimination between DNAs of opposite chirality, as shown previously by Corbett et al. (11). Given that the activity of ParC(ΔCTD) Topo IV is independent of the substrate, we predicted that the FtsK50C derivatives would have equivalent effects on the relaxation of (+)ve and (−)ve sc DNA by Topo IV. This proved to be the case. FtsK50C now inhibited, rather than stimulated, ParC(ΔCTD) Topo IV relaxation of (+)ve scDNA (Figure 6A) while retaining its ability to inhibit relaxation of (−)ve sc DNA (Figure 6B). FtsK50CK997A no longer stimulated relaxation of (+)ve scDNA, but also had no effect on either the (+)ve or (−)ve sc DNA relaxation activity of ParC(ΔCTD) Topo IV (Figure 6C and D, respectively). The increased concentrations of ParC(ΔCTD) Topo IV required to establish relaxation of the DNA substrates suggest a roughly 80- to 120-fold decrease in activity. This observation is consonant with the demonstration by Stone et al. (33) that relaxation of positive supercoils by Topo IV has an inherent processivity of ∼80 strand passage events, as well as with the observation of Neuman et al. (33) that relaxation of negative supercoils is perfectly distributive. Because ParC(ΔCTD) Topo IV can no longer discriminate topology, its activity is expected to be distributive [this is apparent in the data of Corbett et al. (11)]. We suggest that because of this change in activity, ParC(ΔCTD) Topo IV can no longer establish a stimulatory complex with either FtsK50C or FtsK50CK997A and that this destabilized ParC(ΔCTD) Topo IV can be displaced by translocating wild-type FtsK50C.Figure 6.

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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