Probing transient excited states of the bacterial cell division regulator MinE by relaxation dispersion NMR spectroscopy.

Bacterial MinD and MinE form a standing oscillatory wave which positions the cell division inhibitor MinC, that binds MinD, everywhere on the membrane except at the midpoint of the cell, ensuring midcell positioning of the cytokinetic septum. During this process MinE undergoes fold switching as it interacts with different partners. We explore the exchange dynamics between major and excited states of the MinE dimer in 3 forms using N relaxation dispersion NMR: the full-length protein (6-stranded β-sheet sandwiched between 4 helices) representing the resting state; a 10-residue N-terminal deletion (Δ10) mimicking the membrane-binding competent state where the N-terminal helix is detached to interact with membrane; and N-terminal deletions of either 30 (Δ30) or 10 residues with an I24N mutation (Δ10/I24N), in which the β1-strands at the dimer interface are extruded and available to bind MinD, leaving behind a 4-stranded β-sheet.

Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD.

The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo.

Differential affinities of MinD and MinE to anionic phospholipid influence Min patterning dynamics in vitro.

The E. coli Min system forms a cell-pole-to-cell-pole oscillator that positions the divisome at mid-cell. The MinD ATPase binds the membrane and recruits the cell division inhibitor MinC.

MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition.

MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB's poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB.

ParA-mediated plasmid partition driven by protein pattern self-organization.

DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low-copy plasmids, such as the plasmids P1 and F, employ a three-component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker-type ATPase, typically called ParA, which also binds non-specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP-driven patterning is involved in partition is unknown.

DNA requirements for assembly and stability of HIV-1 intasomes.

Integration of viral DNA into the host genome is an essential step in retroviral replication that is mediated by a stable nucleoprotein complex comprising a tetramer of integrase bridging the two ends of the viral DNA in a stable synaptic complex (SSC) or intasome. Assembly of HIV-1 intasomes requires several hundred base pairs of nonspecific internal DNA in addition to the terminal viral DNA sequence that is protected in footprinting experiments. We find that only one of the viral DNA ends in the intasome requires long-nonspecific internal DNA for intasome assembly.

ATP control of dynamic P1 ParA-DNA interactions: a key role for the nucleoid in plasmid partition.

P1 ParA is a member of the Walker-type family of partition ATPases involved in the segregation of plasmids and bacterial chromosomes. ATPases of this class interact with DNA non-specifically in vitro and colocalize with the bacterial nucleoid to generate a variety of reported patterns in vivo. Here, we directly visualize ParA binding to DNA using total internal reflection fluorescence microscopy.

Barrier-to-autointegration factor (BAF) condenses DNA by looping.

Barrier-to-autointegration factor (BAF) is a protein that has been proposed to compact retroviral DNA, making it inaccessible as a target for self-destructive integration into itself (autointegration). BAF also plays an important role in nuclear organization. We studied the mechanism of DNA condensation by BAF using total internal reflection fluorescence microscopy.

Control of transposase activity within a transpososome by the configuration of the flanking DNA segment of the transposon.

The multiple steps of DNA transposition take place within a large complex called the transpososome, in which a pair of transposon DNA ends are synapsed by a multimer of the transposase protein. The final step, a DNA strand transfer reaction that joins the transposon ends to the target DNA strands, entails no net change in the number of high-energy chemical bonds. Physiology demands that, despite remaining stably associated with the transpososome, the strand transfer products undergo neither the reverse reaction nor any further cleavage reactions.

DNA transposition target immunity and the determinants of the MuB distribution patterns on DNA.

MuB, an ATP-dependent DNA-binding protein, is critical for the selection of target sites on the host chromosome during the phage Mu transposition. We developed a multichannel fluidic system to study the MuB-DNA interaction dynamics at the single DNA molecule level by total internal reflection fluorescence microscopy. We analyzed the distribution of MuB along DNA during the assembly and disassembly of MuB polymers on immobilized DNA molecules.

Retroviral DNA integration: reaction pathway and critical intermediates.

The key DNA cutting and joining steps of retroviral DNA integration are carried out by the viral integrase protein. Structures of the individual domains of integrase have been determined, but their organization in the active complex with viral DNA is unknown. We show that HIV-1 integrase forms stable synaptic complexes in which a tetramer of integrase is stably associated with a pair of viral DNA ends.