Limits...
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.

Show MeSH

Related in: MedlinePlus

Stimulation of Topo IV activity is not a result of either DNA translocation or alteration of DNA topology by FtsK50C. (A) The FtsK50C K997A variant has no detectable ATPase activity. Reaction mixtures containing [α-32P]ATP, 5 nM (−)ve sc DNA, the indicated amount of FtsK50C or FtsK50C K997A were incubated for 3 min at 37°C and analyzed as described under ‘Experimental Procedures’ section. The results of three independent experiments were averaged. (B and C) An ATPase mutant of FtsK50C stimulates both Topo IV-catalyzed decatenation and (+)ve scDNA relaxation. Reaction mixtures containing either no Topo IV or 0.05 nM Topo IV, the indicated amounts of FtsK50CK997A, 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 3.8, 7.5, 15 and 60 nM FtsK50CK997A stimulated decatenation by 2.5-, 2.8-, 3.5- and 3.2-fold, respectively (B). Addition of 15 and 60 nM of FtsK50CK997A stimulated relaxation of (+)ve scDNA by 3- and 2.4-fold, respectively (C). (D) Neither the binding of FtsK50C nor of FtsK50CK997A affects either the writhe or the twist of DNA. Reaction mixtures containing either no or the indicated amounts of either FtsK50C or FtsK50CK997A were incubated with relaxed (+)ve scDNA in the presence of AMP-PNP for 30 min as described under ‘Experimental Procedures’ section. Vaccinia virus Topo I was then added to remove unconstrained supercoils. DNA products were analyzed as described under ‘Experimental Procedures’ section. The DNA substrate is shown in the left-most lane of the gel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2875013&req=5

Figure 2: Stimulation of Topo IV activity is not a result of either DNA translocation or alteration of DNA topology by FtsK50C. (A) The FtsK50C K997A variant has no detectable ATPase activity. Reaction mixtures containing [α-32P]ATP, 5 nM (−)ve sc DNA, the indicated amount of FtsK50C or FtsK50C K997A were incubated for 3 min at 37°C and analyzed as described under ‘Experimental Procedures’ section. The results of three independent experiments were averaged. (B and C) An ATPase mutant of FtsK50C stimulates both Topo IV-catalyzed decatenation and (+)ve scDNA relaxation. Reaction mixtures containing either no Topo IV or 0.05 nM Topo IV, the indicated amounts of FtsK50CK997A, 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 3.8, 7.5, 15 and 60 nM FtsK50CK997A stimulated decatenation by 2.5-, 2.8-, 3.5- and 3.2-fold, respectively (B). Addition of 15 and 60 nM of FtsK50CK997A stimulated relaxation of (+)ve scDNA by 3- and 2.4-fold, respectively (C). (D) Neither the binding of FtsK50C nor of FtsK50CK997A affects either the writhe or the twist of DNA. Reaction mixtures containing either no or the indicated amounts of either FtsK50C or FtsK50CK997A were incubated with relaxed (+)ve scDNA in the presence of AMP-PNP for 30 min as described under ‘Experimental Procedures’ section. Vaccinia virus Topo I was then added to remove unconstrained supercoils. DNA products were analyzed as described under ‘Experimental Procedures’ section. The DNA substrate is shown in the left-most lane of the gel.

Mentions: FtsK is an exceptionally fast DNA translocase [∼6 kb/s; (24,25)]. Positive supercoils accumulate ahead of the translocating enzyme, whereas negative supercoils accumulate behind it (18). We have suggested previously that the observed stimulation of Topo IV activity might result from FtsK translocation, generating a preferred substrate in the vicinity of the topoisomerase (14). We reasoned that if this were the case, removing the motor activity of FtsK50C should abolish the observed stimulation. Because FtsK50C uses the energy of ATP hydrolysis to translocate along the DNA (18,25), we purified a Walker A (K997A) mutant of FtsK50C tagged with six His residues at the N-terminus. This variant FtsK has no detectable ATPase activity (Figure 2A) and thus, based on previous studies (18,25), cannot translocate on DNA. Surprisingly, both the decatenation and (+)ve scDNA relaxation activities of Topo IV were stimulated by the presence of FtsK50CK997A to the same extent as by the wild-type protein (Figure 2B and C). We conclude that the observed stimulation is likely to be independent of FtsK translocation.Figure 2.


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

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

Stimulation of Topo IV activity is not a result of either DNA translocation or alteration of DNA topology by FtsK50C. (A) The FtsK50C K997A variant has no detectable ATPase activity. Reaction mixtures containing [α-32P]ATP, 5 nM (−)ve sc DNA, the indicated amount of FtsK50C or FtsK50C K997A were incubated for 3 min at 37°C and analyzed as described under ‘Experimental Procedures’ section. The results of three independent experiments were averaged. (B and C) An ATPase mutant of FtsK50C stimulates both Topo IV-catalyzed decatenation and (+)ve scDNA relaxation. Reaction mixtures containing either no Topo IV or 0.05 nM Topo IV, the indicated amounts of FtsK50CK997A, 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 3.8, 7.5, 15 and 60 nM FtsK50CK997A stimulated decatenation by 2.5-, 2.8-, 3.5- and 3.2-fold, respectively (B). Addition of 15 and 60 nM of FtsK50CK997A stimulated relaxation of (+)ve scDNA by 3- and 2.4-fold, respectively (C). (D) Neither the binding of FtsK50C nor of FtsK50CK997A affects either the writhe or the twist of DNA. Reaction mixtures containing either no or the indicated amounts of either FtsK50C or FtsK50CK997A were incubated with relaxed (+)ve scDNA in the presence of AMP-PNP for 30 min as described under ‘Experimental Procedures’ section. Vaccinia virus Topo I was then added to remove unconstrained supercoils. DNA products were analyzed as described under ‘Experimental Procedures’ section. The DNA substrate is shown in the left-most lane of the gel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875013&req=5

Figure 2: Stimulation of Topo IV activity is not a result of either DNA translocation or alteration of DNA topology by FtsK50C. (A) The FtsK50C K997A variant has no detectable ATPase activity. Reaction mixtures containing [α-32P]ATP, 5 nM (−)ve sc DNA, the indicated amount of FtsK50C or FtsK50C K997A were incubated for 3 min at 37°C and analyzed as described under ‘Experimental Procedures’ section. The results of three independent experiments were averaged. (B and C) An ATPase mutant of FtsK50C stimulates both Topo IV-catalyzed decatenation and (+)ve scDNA relaxation. Reaction mixtures containing either no Topo IV or 0.05 nM Topo IV, the indicated amounts of FtsK50CK997A, 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 3.8, 7.5, 15 and 60 nM FtsK50CK997A stimulated decatenation by 2.5-, 2.8-, 3.5- and 3.2-fold, respectively (B). Addition of 15 and 60 nM of FtsK50CK997A stimulated relaxation of (+)ve scDNA by 3- and 2.4-fold, respectively (C). (D) Neither the binding of FtsK50C nor of FtsK50CK997A affects either the writhe or the twist of DNA. Reaction mixtures containing either no or the indicated amounts of either FtsK50C or FtsK50CK997A were incubated with relaxed (+)ve scDNA in the presence of AMP-PNP for 30 min as described under ‘Experimental Procedures’ section. Vaccinia virus Topo I was then added to remove unconstrained supercoils. DNA products were analyzed as described under ‘Experimental Procedures’ section. The DNA substrate is shown in the left-most lane of the gel.
Mentions: FtsK is an exceptionally fast DNA translocase [∼6 kb/s; (24,25)]. Positive supercoils accumulate ahead of the translocating enzyme, whereas negative supercoils accumulate behind it (18). We have suggested previously that the observed stimulation of Topo IV activity might result from FtsK translocation, generating a preferred substrate in the vicinity of the topoisomerase (14). We reasoned that if this were the case, removing the motor activity of FtsK50C should abolish the observed stimulation. Because FtsK50C uses the energy of ATP hydrolysis to translocate along the DNA (18,25), we purified a Walker A (K997A) mutant of FtsK50C tagged with six His residues at the N-terminus. This variant FtsK has no detectable ATPase activity (Figure 2A) and thus, based on previous studies (18,25), cannot translocate on DNA. Surprisingly, both the decatenation and (+)ve scDNA relaxation activities of Topo IV were stimulated by the presence of FtsK50CK997A to the same extent as by the wild-type protein (Figure 2B and C). We conclude that the observed stimulation is likely to be independent of FtsK translocation.Figure 2.

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.

Show MeSH
Related in: MedlinePlus