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. The viral DNA is processed within these complexes, which go on to capture the target DNA and integrate the viral DNA ends. The joining of the two viral DNA ends to target DNA occurs sequentially, with a stable intermediate complex in which only one DNA end is joined. The integration product also remains stably associated with integrase and likely requires disassembly before completion of the integration process by cellular enzymes. The results define the series of stable nucleoprotein complexes that mediate retroviral DNA integration.

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. This activity depends on, and is highly specific for ATP. DNA-binding activity is not coupled to ATP hydrolysis. Rather, ParA undergoes a slow multi-step conformational transition upon ATP binding, which licenses ParA to bind non-specific DNA. The kinetics provide a time-delay switch to allow slow cycling between the DNA binding and non-binding forms of ParA. We propose that this time delay, combined with stimulation of ParA's ATPase activity by ParB bound to the plasmid DNA, generates an uneven distribution of the nucleoid-associated ParA, and provides the motive force for plasmid segregation prior to cell division.

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. Here we studied Min protein patterning at limiting MinD concentrations reflecting the in vivo conditions. We identified "burst" patterns-radially expanding and imploding binding zones of MinD, accompanied by a peripheral ring of MinE. Bursts share several features with the in vivo dynamics of the Min system including standing wave oscillations. Our data support a patterning mechanism whereby the MinD-to-MinE ratio on the membrane acts as a toggle switch: recruiting and stabilizing MinD on the membrane when the ratio is high and releasing MinD from the membrane when the ratio is low. Coupling this toggle switch behavior with MinD depletion from the cytoplasm drives a self-organized standing wave oscillator.cell division | subcellular organization | pattern formation | self-organization | intracellular positioning

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. We reconstituted and visualized ParA-mediated plasmid partition inside a DNA-carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB-stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion-ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid. The EMBO Journal (2013Journal ( ) 32, 1238Journal ( -1249Journal ( . doi:10.1038Journal ( / emboj.2013 IntroductionHow is energy used to transport and spatially organize large objects, such as DNA, in a cell? In eukaryotes, a mechanically driven mitotic spindle apparatus separates chromosomes. In bacteria, the most common DNA segregation mechanism employed by chromosomes and plasmids is still unclear. All bacterial chromosomes and most naturally occurring plasmids are of low-copy number, and many employ active segregation (or partition) systems to ensure inheritance. Partition systems in bacteria are minimalistic, involving only three principal components: an NTPase that drives partition, a partition site on the DNA target, and a partition site binding protein that forms a large partition complex on the DNA target. The most prevalent class of partition systems in the microbial world use ParA-type ATPases that carry a deviant Walker-type active site, but the mechanism remains elusive.The plasmids P1 and F are stably maintained in Escherichia coli and their partition systems are paradigms for studying ParA-mediated DNA segregation. The three essential plasmidencoded components are ParA (or F SopA)-the ATPase, ParB (or F SopB)-the partition site binding protein, and parS (or F sopC)-the partition site on the plasmid. ParBs load onto and around their cognate partition site to form partition complexes, which have been observed as punctate foci in vivo by fluorescence microscopy (Hirano et al, 1998;Erdmann et al, 1999;Lim et al, 2005;Adachi et al, 2006;Sengupta et al, 2010). ParA ATPase activity is critical to the partition process (Ebersbach and Gerdes, 2001;Fung et al, 2001;Barilla et al, 2005;Pratto et al, 2008). ParA alone has weak ATPase activity that is mildly stimulated by non-specific DNA (nsDNA) or ParB (Davis et al, 1992;Watanabe et al, 1992). Together, ParB and DNA synergistically stimulate ParA ATPase activity. But...

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. MinE interacts with and releases MinD (and MinC) from the membrane. The chase of MinD by MinE creates the in vivo oscillator that maintains a low level of the division inhibitor at mid-cell. In vitro reconstitution and visualization of Min proteins on a supported lipid bilayer has provided significant advances in understanding Min patterns in vivo. Here we studied the effects of flow, lipid composition, and salt concentration on Min patterning. Flow and no-flow conditions both supported Min protein patterns with somewhat different characteristics. Without flow, MinD and MinE formed spiraling waves. MinD and, to a greater extent MinE, have stronger affinities for anionic phospholipid. MinD-independent binding of MinE to anionic lipid resulted in slower and narrower waves. MinE binding to the bilayer was also more susceptible to changes in ionic strength than MinD. We find that modulating protein diffusion with flow, or membrane binding affinities with changes in lipid composition or salt concentration, can differentially affect the retention time of MinD and MinE, leading to spatiotemporal changes in Min patterning.

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The Mu A protein binds site-specifically to the ends of Mu DNA. Two blocks of protection against nuclease are seen at the left (L) end; the right (R) end exhibits one continuous block of protection. We interpret the nuclease protection pattern and sequence data as evidence for three Mu A protein binding sites at each end of Mu. Both the L and R ends have one site close to the terminus; each end also has two additional sites that differ in location between the L and R ends. The Mu A protein protection patterns on the L ends of Mu and the closely related phage D108 are, despite many interspersed sequence differences in one of the protected regions, essentially identical. We show that the A proteins of Mu and D108 can function, at different efficiencies, interchangeably on the Mu and D108 L ends in vivo. Purified Mu repressor, in addition to its primary binding in the operator region, also binds less strongly to the Mu ends at the same sites as the Mu A protein. This affinity of Mu repressor for DNA sites recognized by the Mu A protein may play a role as a second level of control of transposition by the repressor.

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