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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 stimulates both Topo IV-catalyzed decatenation and (+)ve scDNA relaxation. (A) FtsK50C interacts with Topo IV. Two and one half pmol of FtsK50C protein and 180 pmol of the indicated proteins were spotted on a nitrocellulose membrane. Immunoblotting was then performed as in Espeli et al. (14). FtsK50C was visualized by ECL western analysis using an anti-FLAG–HRP conjugate antibody. (B and C) Reaction mixtures containing either no Topo IV or 0.05 nM Topo IV, the indicated amounts of FtsK50C, and either form II : form II DNA dimers (B) or (+)ve scDNA (C) were incubated and analyzed as described under ‘Experimental Procedures’ section. Addition of either 15 or 60 nM of FtsK50C stimulated decatenation by 2.2-fold (B) and relaxation of (+)ve scDNA by 3- and 3.3-fold, respectively (C). F I, form I DNA; F II, form II DNA.
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Figure 1: FtsK50C stimulates both Topo IV-catalyzed decatenation and (+)ve scDNA relaxation. (A) FtsK50C interacts with Topo IV. Two and one half pmol of FtsK50C protein and 180 pmol of the indicated proteins were spotted on a nitrocellulose membrane. Immunoblotting was then performed as in Espeli et al. (14). FtsK50C was visualized by ECL western analysis using an anti-FLAG–HRP conjugate antibody. (B and C) Reaction mixtures containing either no Topo IV or 0.05 nM Topo IV, the indicated amounts of FtsK50C, and either form II : form II DNA dimers (B) or (+)ve scDNA (C) were incubated and analyzed as described under ‘Experimental Procedures’ section. Addition of either 15 or 60 nM of FtsK50C stimulated decatenation by 2.2-fold (B) and relaxation of (+)ve scDNA by 3- and 3.3-fold, respectively (C). F I, form I DNA; F II, form II DNA.

Mentions: Previously, we had observed stimulation of the decatenation activity of Topo IV, using a purified FtsK CTD (Domain 3) tagged with the FLAG epitope at the N-terminus (FtsKC) (14). Because this protein lacked amino acids 179–230, which are required for hexamerization (18), high concentrations were necessary to achieve oligomerization and stimulation of Topo IV. In this report, we have used a version of FtsK that includes amino acids 179–230 linked to the N-terminus of Domain 3 that is also tagged with the FLAG epitope (FtsK50C). Although the amino acids 179–230 are necessary to promote the assembly of the hexameric ring structure on the DNA, they can also cause aggregation of the protein. However, our protein preparation was subjected to analytical gel filtration and no protein aggregates were observed (data not shown). As we have shown for FtsKC (14), FtsK50C also interacts with the ParC subunit of Topo IV (Figure 1A).Figure 1.


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 stimulates both Topo IV-catalyzed decatenation and (+)ve scDNA relaxation. (A) FtsK50C interacts with Topo IV. Two and one half pmol of FtsK50C protein and 180 pmol of the indicated proteins were spotted on a nitrocellulose membrane. Immunoblotting was then performed as in Espeli et al. (14). FtsK50C was visualized by ECL western analysis using an anti-FLAG–HRP conjugate antibody. (B and C) Reaction mixtures containing either no Topo IV or 0.05 nM Topo IV, the indicated amounts of FtsK50C, and either form II : form II DNA dimers (B) or (+)ve scDNA (C) were incubated and analyzed as described under ‘Experimental Procedures’ section. Addition of either 15 or 60 nM of FtsK50C stimulated decatenation by 2.2-fold (B) and relaxation of (+)ve scDNA by 3- and 3.3-fold, respectively (C). F I, form I DNA; F II, form II DNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: FtsK50C stimulates both Topo IV-catalyzed decatenation and (+)ve scDNA relaxation. (A) FtsK50C interacts with Topo IV. Two and one half pmol of FtsK50C protein and 180 pmol of the indicated proteins were spotted on a nitrocellulose membrane. Immunoblotting was then performed as in Espeli et al. (14). FtsK50C was visualized by ECL western analysis using an anti-FLAG–HRP conjugate antibody. (B and C) Reaction mixtures containing either no Topo IV or 0.05 nM Topo IV, the indicated amounts of FtsK50C, and either form II : form II DNA dimers (B) or (+)ve scDNA (C) were incubated and analyzed as described under ‘Experimental Procedures’ section. Addition of either 15 or 60 nM of FtsK50C stimulated decatenation by 2.2-fold (B) and relaxation of (+)ve scDNA by 3- and 3.3-fold, respectively (C). F I, form I DNA; F II, form II DNA.
Mentions: Previously, we had observed stimulation of the decatenation activity of Topo IV, using a purified FtsK CTD (Domain 3) tagged with the FLAG epitope at the N-terminus (FtsKC) (14). Because this protein lacked amino acids 179–230, which are required for hexamerization (18), high concentrations were necessary to achieve oligomerization and stimulation of Topo IV. In this report, we have used a version of FtsK that includes amino acids 179–230 linked to the N-terminus of Domain 3 that is also tagged with the FLAG epitope (FtsK50C). Although the amino acids 179–230 are necessary to promote the assembly of the hexameric ring structure on the DNA, they can also cause aggregation of the protein. However, our protein preparation was subjected to analytical gel filtration and no protein aggregates were observed (data not shown). As we have shown for FtsKC (14), FtsK50C also interacts with the ParC subunit of Topo IV (Figure 1A).Figure 1.

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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