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K Mizuuchi

Publications and source records attributed to K Mizuuchi.

At least 37 records · Page 2Linked to original sources
PubMed1995

Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration.

The crystal structure of the core domain of bacteriophage Mu transposase, MuA, has been determined at 2.4 A resolution. The first of two subdomains contains the active site and, despite very limited sequence homology, exhibits a striking similarity to the core domain of HIV-1 integrase, which carries out a similar set of biochemical reactions. It also exhibits more limited similarity to other nucleases, RNase H and RuvC. The second, a beta barrel, connects to the first subdomain through several contacts. Three independent determinations of the monomer structure from two crystal forms all show the active site held in a similar, apparently inactive configuration. The enzymatic activity of MuA is known to be activated by formation of a DNA-bound tetramer of the protein. We propose that the connections between the two subdomains may be involved in the cross-talk between the active site and the other domains of the transposase that controls the activity of the protein.

Amino Acid Sequence↗
PubMed1994

A novel class of winged helix-turn-helix protein: the DNA-binding domain of Mu transposase.

BACKGROUND: Mu transposase (MuA) is a multidomain protein encoded by the bacteriophage Mu genome. It is responsible for translocation of the Mu genome, which is the largest and most efficient transposon known. While the various domains of MuA have been delineated by means of biochemical methods, no data have been obtained to date relating to its tertiary structure. RESULTS: We have solved the three-dimensional solution structure of the DNA-binding domain (residues 1-76; MuA76) of MuA by multidimensional heteronuclear NMR spectroscopy. The structure consists of a three-membered alpha-helical bundle buttressed by a three-stranded antiparallel beta-sheet. Helices H1 and H2 and the seven-residue turn connecting them comprise a helix-turn-helix (HTH) motif. In addition, there is a long nine-residue flexible loop or wing connecting strands B2 and B3 of the sheet. NMR studies of MuA76 complexed with a consensus DNA site from the internal activation region of the Mu genome indicate that the wing and the second helix of the HTH motif are significantly perturbed upon DNA binding. CONCLUSIONS: While the general appearance of the DNA-binding domain of MuA76 is similar to that of other winged HTH proteins, the connectivity of the secondary structure elements is permuted. Hence, the fold of MuA76 represents a novel class of winged HTH DNA-binding domain.

Bacteriophage mu↗
PubMed1993

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.

Bacteriophage phi X 174↗
PubMed1992

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.

Bacteriophage mu↗
PubMed1992

DNA-promoted assembly of the active tetramer of the Mu transposase.

A stable tetramer of the Mu transposase (MuA) bound to the ends of the Mu DNA promotes recombination. Assembly of this active protein-DNA complex from monomers of MuA requires an intricate array of MuA protein-binding sites on supercoiled DNA, divalent metal ions, and the Escherichia coli HU protein. Under altered reaction conditions, many of these factors stimulate assembly of the MuA tetramer but are not essential, allowing their role in formation of the complex to be analyzed. End-type MuA-binding sites and divalent metal ions are most critical and probably promote a conformational change in MuA that is necessary for multimerization. Multiple MuA-binding sites on the DNA contribute synergistically to tetramer formation. DNA superhelicity assists cooperativity between the sites on the two Mu DNA ends if they are properly oriented. HU specifically promotes assembly involving the left end of the Mu DNA. In addition to dissecting the assembly pathway, these data demonstrate that the tetrameric conformation is intrinsic to MuA and constitutes the form of the protein active in catalysis.

Base Sequence↗
PubMed1991

HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer.

Retroviral DNA integration involves a coordinated set of DNA cutting and joining reactions. Linear viral DNA is cleaved at each 3' end to generate the precursor ends for integration. The resulting recessed 3' ends are inserted into target DNA by a subsequent DNA strand transfer reaction. Purified HIV-1 integration protein carries out both of these steps in vitro. Two novel forms of the dinucleotide cleaved from HIV-1 DNA were identified and one, a cyclic dinucleotide, was used to analyze the stereochemical course of viral DNA cleavage. Both viral DNA cleavage and DNA strand transfer display inversion at chiral phosphorothioates during the course of the reaction. These results suggest that both reactions occur by a one-step mechanism without involvement of a covalent protein-DNA intermediate.

Alkaline Phosphatase↗
PubMed1991

DNase protection analysis of the stable synaptic complexes involved in Mu transposition.

Several critical steps in phage Mu transposition involve specialized protein-DNA complexes. Cleavage of Mu donor DNA by MuA protein leads to the formation of the stable cleaved donor complex (CDC), in which the two Mu DNA ends are held together by MuA. In the subsequent strand-transfer reaction the CDC attacks a target DNA to generate the strand-transfer complex, in which the donor and the target DNAs are covalently joined. We have carried out DNase I protection experiments on these protein-DNA complexes and found that only three MuA binding sites (L1, R1, and R2 of the six total) at the two Mu ends are stably bound by MuA to maintain the paired Mu end structure. The protection extends beyond the ends of the Mu sequence for different lengths (7-20 nucleotides) depending on the strand and the type of complex. After formation of the CDC, the other MuA binding sites (L2, L3, and R3) and internal activation sequence become dispensable for the subsequent strand-transfer reaction.

Bacteriophage mu↗
PubMed1991

Inversion of the phosphate chirality at the target site of Mu DNA strand transfer: evidence for a one-step transesterification mechanism.

Central to transposition of phage Mu are two reactions mediated by the MuA protein. First, MuA introduces single-stranded cuts at the ends of the Mu DNA to generate 3' OH termini. In the subsequent strand-transfer step, the MuA-Mu DNA end complex cuts a target DNA and joins the Mu 3' ends to the 5' ends of the target. DNA containing chiral phosphorothioates was used to demonstrate inversion of the chirality during the course of strand transfer. This result strongly supports a one-step transesterification mechanism in which the 3' OH of the cleaved donor DNA is the attacking nucleophile. Furthermore, this donor 3' OH group was essential for target DNA cleavage. In contrast, during lambda integration the phosphate chirality was retained, as expected for a two-step transesterification involving a covalent protein-DNA intermediate.

Bacteriophage lambda↗
PubMed1991

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.

Allosteric Regulation↗
PubMed1991

A rapid in vitro assay for HIV DNA integration.

Retroviruses synthesize a double stranded DNA copy of their RNA genome after infection of a permissive cell and subsequent integration of this DNA copy into the host genome is necessary for normal viral replication. Integration occurs by a specialized DNA recombination reaction, mediated by the viral IN protein. Because this reaction has no known cellular counterpart, it is a particularly attractive target in the search for specific inhibitors with low toxicity that may serve as therapeutic antiviral agents. We present a simple assay system that is suitable for screening potential inhibitors of HIV DNA integration. Only short oligonucleotides matching one end of HIV DNA and purified HIV IN protein are required as substrates. Furthermore, since each step of the assay can be carried out in the wells of microtiter plates, large numbers of reactions can be processed simultaneously.

Animals↗
PubMed1991

Steady-state kinetic analysis of ATP hydrolysis by the B protein of bacteriophage mu. Involvement of protein oligomerization in the ATPase cycle.

The DNA strand-transfer reaction of bacteriophage Mu requires Mu B protein and ATP for high efficiency. These factors facilitate the capture of target DNA by the donor protein-DNA complex. To understand the mechanism of the Mu B ATPase cycle in the Mu DNA strand-transfer reaction, we undertook a steady-state kinetic analysis of Mu B ATPase. The results reveal complex properties of the ATPase activity; Mu B protein oligomerizes in the presence of ATP, and ATP hydrolysis by the Mu B ATPase is stimulated by the protein oligomerization and shows a positive cooperativity with respect to ATP concentration. Mu B ATPase activity is also modulated by DNA and Mu A protein. DNA alone suppresses the catalytic activity of Mu B ATPase, whereas DNA enhances the apparent binding affinity for ATP. In the presence of Mu A protein together with DNA, however, the catalytic activity is greatly stimulated. Based on these results, we propose a working hypothesis in which oligomerization of Mu B protein plays a key role in its ATPase cycle.

Adenosine Triphosphatases↗
PubMed1989

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.

Bacteriophage mu↗
PubMed1989

Interaction of proteins located at a distance along DNA: mechanism of target immunity in the Mu DNA strand-transfer reaction.

DNA molecules carrying a Mu end(s) are inefficient targets in the Mu DNA strand-transfer reaction. This target immunity is due to preferential dissociation of Mu B protein from DNA molecules that have Mu A protein bound to the Mu end; free DNA is a much poorer target than DNA with Mu B protein bound. We show that Mu B protein, which binds nonspecifically to DNA, is immobile once bound. An encounter between Mu A and Mu B proteins, bound some distance apart along DNA, is necessary to facilitate the Mu B dissociation. Experiments which show that DNA without a Mu end can acquire immunity, by catenation to DNA with a Mu end(s), are consistent with a model of Mu A-Mu B interaction by DNA looping, but not by linear movement of protein(s) along DNA.

Chromosome Mapping↗
PubMed1989

V(D)J recombination: a functional definition of the joining signals.

Two conserved DNA sequences serve as joining signals in the assembly of immunoglobulins and T-cell receptors from V-, (D)-, and J-coding segments during lymphoid differentiation. We have examined V(D)J recombination as a function of joining signal sequence. Plasmid substrates with mutations in one or both of the heptamer-spacer-nonamer sequences were tested for recombination in a pre-B-cell line active in V(D)J recombination. No signal variant recombines more efficiently than the consensus forms of the joining signals. We find the heptamer sequence to be the most important; specifically, the three bases closest to the recombination crossover site are critical. The nonamer is not as rigidly defined, and it is not important to maintain the five consecutive As that distinguish the consensus nonamer sequence. Both types of signals display very similar sequence requirements and have in common an intolerance for changes in spacer length greater than 1 bp. Although the two signal types share sequence motifs, we find no evidence of a role in recombination for homology between the signals, suggesting that they serve primarily as protein recognition and binding sites.

Base Sequence↗