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K Mizuuchi

Publications and source records attributed to K Mizuuchi.

At least 73 records · Page 4Linked to original sources
PubMed1985

A defined system for the DNA strand-transfer reaction at the initiation of bacteriophage Mu transposition: protein and DNA substrate requirements.

An early step in the transposition of bacteriophage Mu DNA in vitro is a DNA strand-transfer reaction that generates an intermediate DNA structure in which the Mu donor DNA and the target DNA are covalently joined. DNA replication, initiated at the DNA forks in this intermediate, generates a cointegrate product; simple insert products can also be formed from the same intermediate by degradation of a specific segment of the structure, followed by gap repair. This DNA strand-transfer reaction requires ATP, magnesium, the Mu A and Mu B proteins, and a factor supplied by an Escherichia coli cell extract. We have now shown that the host protein factor requirement can be satisfied by purified protein HU. The defined system has been used to determine the DNA substrate requirements for the reaction. The reaction requires the two Mu ends, located on the same DNA molecule, in the same relative orientation to one another as in the phage Mu genome. To participate in the strand-transfer reaction efficiently the mini-Mu plasmid, used as the transposon donor, must be supercoiled; the target DNA molecule may be supercoiled, relaxed circular, or linear.

Bacterial Proteins↗
PubMed1984

DNA sequence and transcription of the region upstream of the E. coli gyrB gene.

We have determined the sequence of a 1498 base-pair region in E. coli that extends from within dnaN through recF and into the gyrB gene. An open reading frame of 1071 base pairs has been identified with the recF structural gene. By S1 mapping, we have located a transcription start point 31 base pairs upstream of gyrB. The amount of this transcript is much greater in cells that have been treated with novobiocin, a treatment which is known to induce greater synthesis of DNA gyrase.

Amino Acid Sequence↗
PubMed1984

Cloning and simplified purification of Escherichia coli DNA gyrase A and B proteins.

We have transferred the Escherichia coli gyrA and gyrB genes onto plasmids that allow the overproduction of the DNA gyrase A and B proteins and have designed relatively simple purification procedures for both proteins. The pure proteins are obtained in good yield; from 2 liters of culture (12 g of cells), one can recover 25 mg of GyrA or 3 mg of GyrB protein.

Cloning, Molecular↗
PubMed1984

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.

Base Sequence↗
PubMed1984

Mechanism of transposition of bacteriophage Mu: polarity of the strand transfer reaction at the initiation of transposition.

The distribution of newly synthesized DNA strands in the transposition products of bacteriophage Mu made in an in vitro system has been analyzed. The results support a model in which all Mu transpositions are initiated by a pair of strand transfer reactions that attach the 3' ends of Mu DNA to 5' protruding staggered ends of the target DNA. Joining of these ends produces a pair of structures similar to replication forks at the ends of the Mu DNA. Successful initiation of replication at either one or both ends, followed by a round of semiconservative replication, results in formation of a cointegrate structure. When the intermediate structure fails to replicate, breakage of the junctions between the Mu sequence and the vector sequence derived from the donor molecule can lead to a simple insert with a pair of gaps at the 5' ends of the Mu DNA. Evidence for a gap repair process that completes the simple insertion process has been obtained.

Base Sequence↗
PubMed1983

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.

Bacteriophage mu↗
PubMed1983

Slow cruciform transitions in palindromic DNA.

Extrusion of cruciform structures in self-complementary regions of DNA is known to be favored by negative supercoiling of DNA. We show here that, in moderately supercoiled DNA, cruciform extrusion is a very slow process. In plasmid pUC7 DNA, with a 48-base-pair palindrome, the half-time of extrusion at 50 degrees C is typically several hours; rates are even slower at lower temperature. The rates increase significantly with increasing DNA supercoiling but are only slightly faster in DNA species with much longer palindromes. The reabsorption of cruciform arms is also very slow. The equilibrium between cruciform and regular DNA structures is sensitive to changes in the linking number. Measurement of this equilibrium leads to an estimate of 18 kcal/mol (75.3 kJ/mol) for the free energy required to generate a cruciform structure. In bacterial cells, cruciform DNA may be rare, even when it is thermodynamically favored, because of its slow formation.

Animals↗
PubMed1982

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.

Bacteriophage lambda↗
PubMed1981

Site-specific interaction of DNA gyrase with DNA.

DNA gyrase, in the presence of the inhibitor oxolinic acid, can induce double-strand DNA breakage at specific sites. The sequences at several sites have been determined. In addition, the structure of complexes formed between DNA gyrase and restriction fragments containing an oxolinic acid-promoted cleavage site has been examined by DNase protection methods. DNA gyrase protects more than 120 base pairs of DNA against pancreatic DNase in a region surrounding the cleavage site. Protection is observed both in the presence and absence of oxolinic acid. Protected DNA flanking the cleavage site contains DNase I-sensitive sites spaced on the average 10 or 11 base pairs apart. This result supports the view that, in the DNA gyrase--DNA complex, the DNA is largely wrapped on the outside of the enzyme.

Adenosine Triphosphate↗
PubMed1981

Strand exchange in lambda integrative recombination: genetics, biochemistry, and models.

We have asked, "What is the mechanism of strand exchange during site-specific recombination of phage lambda?" Crosses carried out in vivo have shown that the recombination joint can be extended rather than flush and that the four-strand breaks and rejoinings needed to from a recombinant can occur asynchronously. Crosses carried out in vitro have shown that all the nucleotides at the site of crossover are conserved during recombination, as are most or all of the superhelical turns present in the substrate molecules. We have presented new data showing that topoisomerase activity of Int protein relaxes DNA by making transient single-strand, rather than double-strand, breaks in the phosphodiester back-bone. These findings are incorporated into a model for strand exchange that has as its central intermediate a four-strand structure.

Bacteriophage lambda↗
PubMed1980

DNA gyrase action involves the introduction of transient double-strand breaks into DNA.

DNA gyrase from Escherichia coli, in the presence of ATP, can both separate catenated DNA circles and unknot knotted DNA. Both these reactions require passage of a DNA segment through a transient double-strand break in DNA. Evidence that transient double-strand breaks are also involved in the supercoiling and relaxing activities of DNA gyrase is derived from experiments showing that the linking number of circular DNA is changed in steps of two. A mechanism is proposed for the action of the enzyme.

Adenosine Triphosphate↗