Biomedical subjects

Yasuo Asami

Publications and source records attributed to Yasuo Asami.

5 recordsLinked to original sources

PubMed2007

The Si-tag for immobilizing proteins on a silica surface.

Targeting functional proteins to specific sites on a silicon device is essential for the development of new biosensors and supramolecular assemblies. Using intracellular lysates of several bacterial strains, we found that ribosomal protein L2 binds tightly to silicon particles, which have surfaces that are oxidized to silica. A fusion of E. coli L2 and green fluorescence protein adsorbed to the silica particles with a K(d) of 0.7 nM at pH 7.5 and also adsorbed to glass slides. This fusion protein was retained on the glass slide even after washing for 24 h with a buffer containing 1 M NaCl. We mapped the silica-binding domains of E. coli L2 to amino acids 1-60 and 203-273. These two regions seemed to cooperatively mediate the strong silica-binding characteristics of L2. A fusion of L2 and firefly luciferase also adsorbed on the glass slide. This L2 silica-binding tag, which we call the "Si-tag," can be used for one-step targeting of functional proteins on silica surfaces.

Bacterial Proteins↗

PubMed2006

Visualization of the interaction between archaeal DNA polymerase and uracil-containing DNA by atomic force microscopy.

Deamination of cytosine to uracil is a hydrolytic reaction that is greatly accelerated at high temperatures. The resulting uracil pairs with adenine during DNA replication, thereby inducing G:C to A:T transitions in the progeny. Interestingly, B-family DNA polymerases from hyperthermophilic Archaea recognize the presence of uracil in DNA and stall DNA synthesis. To better understand the recognition mechanism, the binding modes of DNA polymerase B1 of Sulfolobus solfataricus (Pol B1) to uracil-containing DNA were examined by gel mobility shift assays and atomic force microscopy. Although PolB1 per se specifically binds to uracil-containing single-stranded DNA, the binding efficiency was substantially enhanced by the initiation of DNA synthesis. Analysis by the atomic force microscopy showed a number of double-stranded DNA (dsDNA) in the products of DNA synthesis. The generation of ds DNA was significantly inhibited, however, by the presence of template uracil, and intermediates where monomeric forms of Pol B1 appeared to bind to uracil-containing DNA were observed. These results suggest that Pol B1 more efficiently recognizes uracil in DNA during DNA synthesis rather than during random diffusion in solution, and that single molecules of Pol B1 bind to template uracil and stall DNA synthesis.

DNA↗

PubMed2003

Type I restriction enzyme with RecA protein promotes illegitimate recombination.

Illegitimate (non-homologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs. However, we have found a type of illegitimate recombination that requires an interaction between long homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type I (EcoKI) restriction in vivo within a special mutant strain of Escherichia coli. In the present work, we analyzed genetic requirements for this type of illegitimate recombination in well-defined genetic backgrounds. Our analysis demonstrated dependence on RecA function and on the presence of two EcoKI sites on the substrate DNA. These results are in harmony with a model in which EcoKI restriction enzyme attacks an intermediate of homologous recombination to divert it to illegitimate recombination.

Base Sequence↗

PubMed2003

Multiplication of a restriction-modification gene complex.

Previous works have suggested that some gene complexes encoding a restriction (R) enzyme and a cognate modification (M) enzyme may behave as selfish mobile genetic elements. RM gene complexes, which destroy 'non-self' elements marked by the absence of proper methylation, are often associated with mobile genetic elements and are involved in various genome rearrangements. Here, we found amplification of a restriction-modification gene complex. BamHI gene complex inserted into the Bacillus chromosome showed resistance to replacement by a homologous stretch of DNA. Some cells became transformed with the donor without losing BamHI. In most of these transformants, multiple copies of BamHI and the donor allele were arranged as tandem repeats. When a clone carrying one copy of each allele was propagated, extensive amplification of BamHI and the donor unit was observed in a manner dependent on restriction enzyme gene. This suggests that restriction cutting of the genome participates in the amplification. Visualization by fluorescent in situ hybridization revealed that the amplification occurred in single cells in a burst-like fashion that is reminiscent of induction of provirus replication. The multiplication ability in a bacterium with natural capacity for DNA release, uptake and transformation will be discussed in relation to spreading of RM gene -complexes.

Bacillus↗

PubMed2002

Effect of the DNA topoisomerase II inhibitor VP-16 on illegitimate recombination in yeast chromosomes.

Etoposide and teniposide, derivatives of podophyllotoxin, are inhibitors of DNA topoisomerase II and are potent anticancer agents. An adverse effect linked to the use of these drugs is the development of acute myeloid leukemia, a disorder usually associated with chromosomal translocation. To examine podophyllotoxin-induced DNA rearrangement, we developed an assay system to measure illegitimate recombination in Saccharomyces cerevisiae chromosomes. This approach uses juxtaposed CAN1-CYH2 negative selection markers that are introduced into the LEU2 locus, which is located on chromosome III, in a yeast strain carrying the mutated can1 and cyh2 genes. Upon formation of a deletion over the active CAN1-CYH2 genes, a cell becomes resistant to both canavanine and cycloheximide. To introduce drugs into the cell, we used a yeast strain carrying an ISE2 mutation, thereby making the cell drug-permeable. Here we show that treatment of cells with etoposide (VP-16) increases the rate of illegitimate recombination in yeast, indicating that VP-16 stimulates DNA topoisomerase-mediated illegitimate recombination. Structural analysis of the resulting recombinants indicate that most are formed by deletion mutations on chromosome III, which take place between short homologous regions of DNA. We propose a model for illegitimate recombination, in which VP-16 facilitates formation of a cleavable complex between DNA topoisomerase II and DNA, thus promoting DNA double-strand breakage with the resulting DNA ends joined by a non-homologous mechanism.

3-Isopropylmalate Dehydrogenase↗