The MukB-topoisomerase IV interaction mutually suppresses their catalytic activities

Abstract

The bacterial condensin MukB and the cellular chromosomal decatenase, topoisomerase IV interact and this interaction is required for proper condensation and topological ordering of the chromosome. Here, we show that Topo IV stimulates MukB DNA condensation by stabilizing loops in DNA: MukB alone can condense nicked plasmid DNA into a protein–DNA complex that has greater electrophoretic mobility than that of the DNA alone, but both MukB and Topo IV are required for a similar condensation of a linear DNA representing long stretches of the chromosome. Remarkably, we show that rather than MukB stimulating the decatenase activity of Topo IV, as has been argued previously, in stoichiometric complexes of the two enzymes each inhibits the activity of the other: the ParC subunit of Topo IV inhibits the MukF-stimulated ATPase activity of MukB and MukB inhibits both DNA crossover trapping and DNA cleavage by Topo IV. These observations suggest that when in complex on the DNA, Topo IV inhibits the motor function of MukB and the two proteins provide a stable scaffold for chromosomal DNA condensation.

Inter- and intra-Subunit interactions driving the MukBEF ATPase

“…ParC inhibits the MukBF ATPase (21), implying, given the folded conformation of MukB (23,24) and our crosslinking results, that ParC might interpose itself between the MukB hinge and neck regions and that this might disrupt the ATPase cycle, further suggesting that isolated MukB hinge might stimulate the MukBF ATPase in trans . This proved to be the case.…”

“…In order to develop insight into the interactions between MukBEF subunits and the mechanism of inhibition of the MukBF ATPase by the ParC subunit of Topo IV (21), we performed disuccinimldyl glutarate-catalyzed protein-protein crosslinking experiments with MukB alone, MukB and ParC, and MukB and MukF, followed by trypsin digestion and analysis of the cross-linked peptides by mass spectrometry. The crosslinks found between ParC and MukB, MukF and MukB, and selected crosslinks for MukB alone are displayed in Fig.…”

“…MukB and Topo IV are often found in the same locations on the chromosome (18-20). We have shown that when in a stoichiometric complex together, MukB and Topo IV inhibit the catalytic activities of each other: ParC inhibits the ATPase activity of MukB, whereas MukB inhibits DNA transport segment trapping and DNA cleavage by Topo IV (21), suggesting that any ATP-dependent DNA manipulation catalyzed by MukBEF is modulated by Topo IV.…”

Intersubunit and intrasubunit interactions driving the MukBEF ATPase

“…In order to develop insight into the interactions between MukBEF subunits and the mechanism of inhibition of the MukBF ATPase by the ParC subunit of topo IV ( 24 ), we performed di-( N -succinimidyl) glutarate–catalyzed protein–protein crosslinking experiments with MukB alone, MukB and ParC, and MukB and MukF, followed by trypsin digestion and analysis of the cross-linked peptides by mass spectrometry (MS). The crosslinks found between ParC and MukB, MukF and MukB, and selected crosslinks for MukB alone are displayed in Figure 4 .…”

“…MukB and topo IV are often found in the same locations on the chromosome ( 21 , 22 , 23 ). We have shown that when in a stoichiometric complex together, MukB and topo IV inhibit the catalytic activities of each other: ParC inhibits the ATPase activity of MukB, whereas MukB inhibits DNA transport segment trapping and DNA cleavage by topo IV ( 24 ), suggesting that any ATP-dependent DNA manipulation catalyzed by MukBEF is modulated by topo IV.…”