Kiyoshi Mizuuchi

T4 Endonuclease VII Cleaves Holliday Structures

T4 endonuclease VII cleaves Holliday structures in vitro by cutting two strands of the same polarity at or near the branch point. The two unbranched duplexes produced by cleavage each contain a strand break that can be sealed by DNA ligase. This suggests that the cut sites are at the same position in the nucleotide sequence in each strand. The joint action of endonuclease VII and DNA ligase can therefore resolve Holliday structures into genetically sensible products. These observations account for the role of endonuclease VII in the DNA metabolism of phage T4, and provide the first example of an enzyme that acts specifically on branch points in duplex DNA.

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.

In vitro transposition of bacteriophage Mu: A biochemical approach to a novel replication reaction

The transposition-replication reaction of phage Mu has been reproduced in a cell-free reaction system. Two assay methods were used for the detection of transposition products. The first method uses lambda DNA as the target of transposition and a plasmid containing the ends of Mu DNA and an ampicillin-resistance gene as the donor; after the reaction, in vitro lambda packaging allows the scoring of ampr transducing phages generated by transposition. In the second method, the products made in the presence of a radioactive precursor for DNA synthesis are directly analyzed by gel electrophoresis and unique product species are identified. The reaction requires a donor DNA carrying the two Mu ends in their proper relative orientation, extracts containing the A and B gene products of Mu, and host factor(s). RNA synthesis by E. coli RNA polymerase is not required for the reaction. The products include both cointegrates and simple inserts. Both types of products show incorporation of radioactive DNA precursors; however, simple inserts do not seem to undergo a full round of DNA replication.

Role of DNA topology in Mu transposition: Mechanism of sensing the relative orientation of two DNA segments

DNA strand transfer at the initiation of Mu transposition normally requires a negatively supercoiled transposon donor molecule, containing both ends of Mu in inverted repeat orientation. We propose that the specific relative orientation of the Mu ends is needed only to energetically favor a particular configuration that the ends must adopt in a synaptic complex. The model was tested by constructing special donor DNA substrates that, because of their catenation or knotting, energetically favor this same configuration of the Mu ends, even though they are on separate molecules or in direct repeat orientation. These structures are efficient substrates for the strand transfer reaction, whereas appropriate control structures are not. The result eliminates tracking or protein scaffold models for orientation preference. Several other systems in which the relative orientation of two DNA segments is sensed may utilize the same mechanism.

Mechanism of transposition of bacteriophage Mu: structure of a transposition intermediate

Mu transposition works efficiently in vitro and generates both cointegrate and simple insert products. We have examined the reaction products obtained under modified in vitro reaction conditions that do not permit efficient initiation of DNA replication. The major product is precisely the intermediate structure predicted from one of the current models of DNA transposition. Both cointegrates and simple inserts can be made in vitro using this intermediate as the DNA substrate, demonstrating that it is indeed a true transposition intermediate. The requirements for efficient formation of the intermediate include the Mu A protein, the Mu B protein, an unknown number of E. coli host proteins, ATP, and divalent cation. Only E. coli host proteins are required for conversion of the intermediate to cointegrate or simple insert products. Structures resulting from DNA strand transfer at only one end of the transposon are not observed, suggesting that the strand transfers at each end of the transposon are tightly coupled.

Single active site catalysis of the successive phosphoryl transfer steps by DNA transposases: insights from phosphorothioate stereoselectivity.

The transposase family of proteins mediate DNA transposition or retroviral DNA integration via multistep phosphoryl transfer reactions. For Tn10 and phage Mu, a single active site of one transposase protomer catalyzes the successive transposition reaction steps. We examined phosphorothioate stereoselectivity at the scissile position for all four reaction steps catalyzed by the Tn10 transposase. The results suggest that the first three steps required for double-strand cutting at the transposon end proceed as a succession of pseudo-reverse reaction steps while the 3 end of the transposon remains bound to the same side of the active site. However, the mode of substrate binding to the active site changes for the cut transposon 3 end to target DNA strand joining. The phosphorothioate stereoselectivity of the corresponding steps of phage Mu transposition and HIV DNA integration matches that of Tn10 reaction, indicating a common mode of substrate-active site interactions for this class of DNA transposition reactions.

Surfing biological surfaces: Exploiting the nucleoid for partition and transport in bacteria

The ParA family of ATPases is responsible for transporting bacterial chromosomes, plasmids and large protein machineries. ParAs pattern the nucleoid in vivo, but how patterning functions or is exploited in transport is of considerable debate. Here we discuss the process of self‐organization into patterns on the bacterial nucleoid and explore how it relates to the molecular mechanism of ParA action. We review ParA‐mediated DNA partition as a general mechanism of how ATP‐driven protein gradients on biological surfaces can result in spatial organization on a mesoscale. We also discuss how the nucleoid acts as a formidable diffusion barrier for large bodies in the cell, and make the case that the ParA family evolved to overcome the barrier by exploiting the nucleoid as a matrix for movement.

Involvement of supertwisted DNA in integrative recombination of bacteriophage lambda

Abstract Negatively supertwisted closed circular DNA is the primary substrate for integrative recombination of phage λ DNA in vitro . Closed circular λ DNA without supertwists must be converted to the supertwisted form by the action of Escherichia coli DNA gyrase before efficient recombination can occur. When negatively supertwisted substrate is provided, E. coli DNA gyrase and its cofactors are dispensable components of recombination reaction mixtures. In the absence of DNA gyrase activity, circular DNA considerably less negatively twisted than naturally occurring supercoils is an effective substrate, but positively supertwisted DNA appears to be an ineffective substrate. The predominant products of integrative recombination in vitro are covalently closed circles. The closure of the recombined sites appears to occur without appreciable DNA synthesis and without the action of E. coli DNA ligase. No detectable difference can be observed between the degree of supertwisting of product DNA and that of unrecombined DNA. These facts suggest that the resealing of broken DNA strands is an integral part of the recombination reaction mechanism and is closely coupled with the breakage and realignment steps of recombination.