Michiyo Mizuuchi

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. 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.

Cruciform structures in palindromic DNA are favored by DNA supercoiling

Abstract A totally palindromic circular DNA has been prepared by head-to-head ligation of a restriction fragment of plasmid pBR322 DNA. When negatively supercoiled, this DNA readily converts to a cruciform structure, as seen by either electron microscopy or gel electrophoresis. If the DNA is further supercoiled by DNA gyrase after hairpin formation has been initiated, as much as 80% of the molecular length can be extruded into hairpins. The rate of formation of the cruciform structure is strongly temperature-dependent; it is at least five-fold slower at 25 °C than at 35 °C. The palindromic DNA, although it contains all the necessary genetic information, is unable to transform Escherichia coli. We suggest that the intracellular formation of large cruciform structures is incompatible with survival of the DNA species.

Assembly of the active form of the transposase-Mu DNA complex: A critical control point in Mu transposition

Discovery and characterization of a new intermediate in Mu DNA transposition allowed assembly of the transposition machinery to be separated from the chemical steps of recombination. This stable intermediate, which accumulates in the presence of Ca2+, consists of the two ends of the Mu DNA synapsed by a tetramer of the Mu transposase. Within this stable synaptic complex (SSC), the recombination sites are engaged but not yet cleaved. Thus, the SSC is structurally related to both the cleaved donor and strand transfer complexes, but precedes them on the transposition pathway. Once the active protein-DNA complex is constructed, it is conserved throughout transposition. The participation of internal sequence elements and accessory factors exclusively during SSC assembly allows recombination to be controlled prior to the irreversible chemical steps.

Site-specific recognition of the bacteriophage mu ends by the mu a protein

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.

Efficient Mu transposition requires interaction of transposase with a DNA sequence at the Mu operator: Implications for regulation

Phage Mu transposition is initiated by the Mu DNA strand-transfer reaction, which generates a branched DNA structure that acts as a transposition intermediate. A critical step in this reaction is formation of a special synaptic DNA-protein complex called a plectosome. We find that formation of this complex involves, in addition to a pair of Mu end sequences, a third cis-acting sequence element, the internal activation sequence (IAS). The IAS is specifically recognized by the N-terminal domain of Mu transposase (MuA protein). Neither the N-terminal domain of MuA protein nor the IAS is required for later reaction steps. The IAS overlaps with the sequences to which Mu repressor protein binds in the Mu operator region; the Mu repressor directly inhibits the Mu DNA strand-transfer reaction by interfering with the interaction between MuA protein and the IAS, providing an additional mode of regulation by the repressor.

Division of labor among monomers within the Mu transposase tetramer

A single tetramer of Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Analysis of C-terminal deletion derivatives of MuA reveals that a 30 amino acid region between residues 575 and 605 is critical for these three steps. Although inactive on its own, a deletion protein lacking this region assembles with the wild-type protein. These mixed tetramers carry out donor cleavage but do not promote strand transfer, even when the donor cleavage stage is bypassed. These data suggest that the active center of the transposase is composed of the C-terminus of four MuA monomers; one dimer carries out donor cleavage while all four monomers contribute to strand transfer.

MuB protein allosterically activates strand transfer by the transposase of phage Mu

The MuA and MuB proteins collaborate to mediate efficient transposition of the phage Mu genome into many DNA target sites. MuA (the transposase) carries out all the DNA cleavage and joining steps. MuB stimulates strand transfer by activating the MuA-donor DNA complex through direct protein-protein contact. The C-terminal domain of MuA is required for this MuA-MuB interaction. Activation of strand transfer occurs irrespective of whether MuB is bound to target DNA. When high levels of MuA generate a pool of free MuB (not bound to DNA) or when chemical modification of MuB impairs its ability to bind DNA, MuB still stimulates strand transfer. However, under these conditions, intramolecular target sites are used exclusively because of their close proximity to the MuA-MuB-donor DNA complex.

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. 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 ParAs 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.

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. 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.

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. We found that BAF compacts DNA by a looping mechanism. Dissociation of BAF from DNA occurs with multiphasic kinetics; an initial fast phase is followed by a much slower dissociation phase. The mechanistic basis of the broad timescale of dissociation is discussed. This behavior mimics the dissociation of BAF from retroviral DNA within preintegration complexes as monitored by functional assays. Thus the DNA binding properties of BAF may alone be sufficient to account for its association with the preintegration complex.