Yutaka Ito

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2′ methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2′ methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.

Conformation change of effector-region residues in antiparallel β-sheet of human c-Ha-ras protein on GDP→GTPγS exchange: A two-dimensional NMR study

Abstract The conformations of a truncated human c-Ha- ras gene product [ ras (1–171) protein] in the GDP-bound form and in the GTPγS-bound form were compared by two-dimensional nuclear Overhauser effect spectroscopy (NOESY). As for the GDP-bound ras (1–171) protein, three NOESY cross peaks were observed in the region of 4.5–6.0 ppm, indicating a regular antiparallel β-sheet structure. On the ligand exchange from GDP to GTPγS, one of the three NOESY cross peaks disappeared and the other two cross peaks were appreciably shifted. By analysis of the effects of specific deuteration of leucine residues and the homonuclear Hartmann-Hahn spectroscopy, the antiparallel β-sheet was found to consist of residues 38–44 and residues 51–57. The conformations around Ser-39 and Leu-56 are of the regular antiparallel β-strand type in the GDP-bound state, and largely distorted in the GTPγS-bound state, which is probably related to the conformational activation of the effector region of ras proteins by ligand exchange from GDP to GTPγS.

Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution

SummaryAll the backbone 1H and 15N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by 15N-edited two-dimensional NMR experiments on selectively 15N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly 15N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or Cαprotons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and Cα protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six β strands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the β-sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state.

A 1H-15N NMR study of human c-Ha-ras protein: biosynthetic incorporation of 15N-labeled amino acids.

SummaryA1H-15N NMR study was performed on the GDP-bound form of a truncated human c-Ha-ras oncogene product (171 amino acid residues). Resonance cross peaks of the backbone amide1H-15N nuclei of a uniformly15N-labeled protein were observed with heteronuclear-single-quantum coherence spectroscopy (HSQC). In order to resolve overlapping cross peaks, selective15N-labeling of one or two types of amino acid residues (Ala, Arg, Asx, Glx, Gly, His, lie, Leu, Lys, Met, Phe, Ser; Thr, Tyr and/or Val) was carried out using appropriateE. coli mutant strains. By this procedure, all the backbone1H-15N cross peaks were classified into amino acid types.

SENSITIVE GERMANIUM THERMISTORS FOR CRYOGENIC THERMAL DETECTOR OF TOKYO DARK MATTER SEARCH PROGRAMME

Abstract Sensitive n-type and p-type germanium thermistors were fabricated by the melt doping technique and by the neutron transmutation doping (NTD) technique, respectively, aiming at the use for cryogenic thermal detector, or bolometer of Tokyo dark matter search programme. We report on the measurements of the sensitivities of these thermistors. In particular, the p-type thermistors are sensitive enough to scale up our existing prototype LiF bolometer and realize a multiple array of the bolometers with the total absorber mass of about 1 kg.

Cryogenic thermal detector with LiF absorber for direct dark matter search experiment

Abstract A cryogenic thermal detector with a 2.8 g LiF crystal is constructed. A photoelectric peak of 60 keV γ-rays is clearly seen with LiF crystal with an RMS energy resolution of 3.8 keV. LiF is suitable for the detection of possible particle dark matter with a spin-dependent interaction like neutralinos since both Li and F are expected to have high interaction rates with such particles.

Development of the bolometer for the β+β+ decay experiment

Abstract We developed a bolometer with a 0.5 g CdTe absorber using a high-sensitivity NTD germanium thermistor. An energy resolution of 24 keV (FWHM) is obtained for 0.66 to 1.8 MeV γ-rays. It shows a good linearity of the response in the energy range between 0.66 and 5.5 MeV. The gain is found to be stable for a period of 72 days of continuous operation and relatively insensitive to temperature changes of the refrigerator. The temperature change of the refrigerator for this period is within ±1 mK. Since this material contains double positron emission ( β + β + ) nuclides 106 Cd, 108 Cd and 120 Te, a new series of double beta decay experiments may become possible with this new method.

Direct dark matter search program with bolometers at the University of Tokyo

Bolometers are being developed at the University of Tokyo aiming at a search for galactic dark matter. We have succeeded in constructing a small-sized bolometer with 2.8 gram of lithium fluoride crystal with an rms energy resolution of 3.8 ke V for 60-ke V γ rays. Both lithium and fluorine are expected to be suitable for the detection of possible particle dark matter with a spin-dependent interaction like supersymmetric neutralinos. Fabrication of Neutron Transmutation Doped (NTD) germanium is also in progress to make a number of thermistors with uniform sensitivities to be used for multi-module bolometers. When the development of a full-sized detector with a total mass of order one kilogram is completed, we plan to install it in the Kamioka underground laboratory.

Performance of a lithium fluoride bolometer for Tokyo dark matter search experiment

Abstract The performance of a 21-g lithium fluoride bolometer is presented. The background spectrum was measured in the surface laboratory. We derive an exclusion plot for the spin-dependent coupled Weakly Interacting Massive Particles (WIMPs) cross section.

Guanine-nucleotide binding activity, interaction with GTPase-activating protein and solution conformation of the human c-Ha-Ras protein catalytic domain are retained upon deletion of C-terminal 18 amino acid residues.

A trucated human c-Ha-Ras protein that lacks the C-terminal 18 amino acid residues and the truncated Ras protein with the amino acid substitution Gly → Val in position 12 were prepared by anE. coli overexpression system. The truncated Ras protein showed the same guanine-nucleotide binding activity and GTPase activity as those of the full-length Ras protein. Further, the same extent of GTPase activity enhancement due to GTPase-activating protein was observed for the truncated and full-length Ras proteins. In fact, two-dimensional proton NMR analyses indicated that the tertiary structure of the truncated Ras protein (GDP-bound or GMPPNP-bound) was nearly the same as that of the corresponding catalytic domain of the full-length Ras protein. Moreover, a conformational change around the effector region upon GDP → GMPPNP exchange occurred in the same manner for both proteins. These observations indicate that the C-terminal flanking region (18 amino acid residues) of the Ras protein does not appreciably interact with the catalytic domain. Therefore, the truncated Ras protein is suitable for studying the molecular mechanism involved in the GTPase activity and the interaction with the GTPase-activating protein. On the other hand, an active form of the truncated Ras protein, unlike that of the full-length Ras protein, did not induce neurite outgrowth of rat pheochromocytoma PC12 cells. Thus, membrane anchoring of the Ras protein through its C-terminal four residues is not required for the interaction of Ras and GAP, but may be essential for the following binding of the Ras-GAP complex with the putative downstream target.