We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase\lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg\l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a Bd-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with K m and k cat values of 11p2 µM (meanpS.D., n l 3) and 6610p880 s −" (meanpS.D., n l 3)

Inositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO2) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP6 (phytate), its pyrophosphate derivatives InsP7 and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO2 bead purification also enabled us to quantify InsP6 in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid. These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.

A thermophilic and thermostable P-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Suljiulobus sovuturicus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 'C with o-nitrophenyl P-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus P-galactosidase was a tetramer of 240 Ifr S kDa composed of similar or identical subunits. Comparison of the amino acid composition of pgalactosidase from S. solfataricus with that from Eschevichia culi revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not crossreact with P-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for P-galactosidases from other mesopbilic and thermophilic sources. More recently, a new P-galactosidase activity has been identified in E. colicells with LacZ deletion selected for growth on lactose [6]. This enzyme, termed ebg for evolved pgalactosidases, has a subunit molecular mass of 120 kDa, very close to that of the LacZ enzyme. However, the ebg protein has a hexameric and not a tetrameric structure, and the two enzymes are not immunologically related.In recent years, p-galactosidase activities from various microbial sources have been purified and characterized for their physicochemical properties, reaction requirements and substrate specificities [l, 71. Thermostable p-galactosidases [S ~ 1 I] have received considerable attention because of their possible utilization in the industrial processing of lactose-contain-4,51.

The Mini-Chromosome Maintenance (MCM) proteins are candidates of replicative DNA helicase in eukarya and archaea. Here we report a 2.8 Å crystal structure of the N-terminal domain (residues 1–268) of the Sulfolobus solfataricus MCM (Sso MCM) protein. The structure reveals single-hexameric ring-like architecture, at variance from the protein of Methanothermobacter thermoautotrophicus (Mth). Moreover, the central channel in Sso MCM seems significantly narrower than the Mth counterpart, which appears to more favorably accommodate single-stranded DNA than double-stranded DNA, as supported by DNA-binding assays. Structural analysis also highlights the essential role played by the zinc-binding domain in the interaction with nucleic acids and allows us to speculate that the Sso MCM N-ter domain may function as a molecular clamp to grasp the single-stranded DNA passing through the central channel. On this basis possible DNA unwinding mechanisms are discussed.

Mini-chromosome Maintenance (MCM) proteins play an essential role in both initiation and elongation phases of DNA replication in Eukarya. Genes encoding MCM homologs are present also in the genomic sequence of Archaea and the MCM-like protein from the euryarchaeon Methanobacterium thermoautotrophicum (Mth MCM) was shown to possess a robust ATP-dependent 3-5 DNA helicase activity in vitro. Herein, we report the first biochemical characterization of a MCM homolog from a crenarchaeon, the thermoacidophile Sulfolobus solfataricus (Sso MCM). Gel filtration and glycerol gradient centrifugation experiments indicate that the Sso MCM forms single hexamers (470 kDa) in solution, whereas the Mth MCM assembles into double hexamers. The Sso MCM has NTPase and DNA helicase activity, which preferentially acts on DNA duplexes containing a 5-tail and is stimulated by the single-stranded DNA binding protein from S. solfataricus (Sso SSB). In support of this functional interaction, we demonstrated by immunological methods that the Sso MCM and SSB form protein⅐protein complexes. These findings provide the first in vitro biochemical evidence of a physical/functional interaction between a MCM complex and another replication factor and suggest that the two proteins may function together in vivo in important DNA metabolic pathways. Mini-chromosome Maintenance (MCM)1 genes (MCMs 2-7) were originally identified in budding and fission yeast by a genetic analysis of mutants that were unable to efficiently replicate mini-chromosomes (1, 2). Homologs of the six yeast MCM genes were subsequently identified in various other eukaryotic organisms, from Drosophila melanogaster to Homo sapiens and found to code for proteins (ranging in length from 776 to 1017 amino acidic residues), which are evolutionarily conserved especially in the central third of their polypeptide chain (3, 4). In fact, this region contains the four sequence motifs typically found in DNA helicases, including the Walker A and B boxes that are critical for nucleotide binding and hydrolysis (5). The MCM proteins are relatively abundant in proliferating cells and were purified from cell extracts of various organisms either as hetero-hexameric complexes containing all six polypeptides or as sub-assemblies of various subunit composition (such as MCM 2/4/6/7 and MCM 4/6/7 (6 -10)). However, among all these multimeric complexes only the MCM 4/6/7 hexamer was demonstrated to have a weak and nonprocessive DNA helicase activity (11-13). The MCM 4/6/7 complex is dis-assembled in vitro upon addition of MCM 2 or MCM 3/5, and this causes inhibition of its DNA unwinding activity (13,14). Based on these findings, it was proposed that the MCM 4/6/7 assembly could act as DNA unwinding factor at the replication origins, whereas the other MCM subunits could play regulatory functions. However, due to the limited processivity of their DNA unwinding activity the MCM proteins were considered poor candidates for the helicase associated with the DNA replication fork. In addition, several genetic studie...

We report on shadowgraphic measurements showing the first space-and time-resolved snapshots of ultraintense laser pulse-generated fast electrons propagating through a solid target. A remarkable result is the formation of highly collimated jets (,20-mm) traveling at the velocity of light and extending up to 1 mm. This feature clearly indicates a magnetically assisted regime of electron transport, of critical interest for the fast ignitor scheme. Along with these jets, we detect a slower (ഠc͞2) and broader (up to 1 mm) ionization front consistent with collisional hot electron energy transport. 52.60. + h The fast ignitor scheme, which claims to relax some of the constraints hampering the standard approaches to inertial confinement fusion, has triggered a worldwide interest since its inception [1]. It hinges on the rapid additional heating of the core of a precompressed thermonuclear pellet due to the slowing down of a bunch of relativistic electrons generated by an ultraintense laser pulse. Now, the highly overcritical plasma surrounding the core should prevent any laser pulse from reaching it, whatever highintensity penetration mechanisms are at work (relativistic self-induced transparency [2] or ponderomotive hole boring [3]). An encouraging point is that particle-in-cell simulations predict a rather peaked hot electron distribution in the vicinity of the laser-solid interaction zone [4]. However, an efficient heating of the core requires the electron beam to remain collimated up to its final absorption zone, i.e., on a distance of several hundreds of microns. This can be achieved only through the pinching effect of the beam-driven magnetic field competing with multiple scattering. Therefore, fast electron transport from moderately to extremely dense regions appears as a key issue for the success of fast ignition, which must be thoroughly tackled both experimentally and theoretically.Over the past year, there has been a growing body of experimental evidence pointing to the existence of very collimated high intensity laser-produced electron jets traveling through solid targets. Tatarakis et al. have recently observed a narrow expanding plasma at the rear surface of thick plastic slabs irradiated by a 1 ps, 10 19 W͞cm 2 laser pulse [5]. By using a 2D Fokker-Planck hybrid code, they interpreted this localized rear heating as a magnetic field-enhanced electron energy deposition at the target/vacuum interface [6]. This effect has also been detected in other experiments [7]. Though very encouraging, these studies still provide an incomplete experimental picture of the phenomena arising in the bulk of the target.In the present paper, we report on optical shadowgraphic results showing what is, to our knowledge, the first comprehensive set of space-and time-resolved snapshots of fast electrons propagating through a solid target. In order to bypass the classical limitation of optical probing into an overcritical solid target, we use transparent glass slides. Our measurements pinpoint the existence of two types of fast...

Establishment of sister chromatid cohesion is coupled to DNA replication, but the underlying molecular mechanisms are incompletely understood. DDX11 (also named ChlR1) is a super-family 2 Fe-S cluster-containing DNA helicase implicated in Warsaw breakage syndrome (WABS). Herein, we examined the role of DDX11 in cohesion establishment in human cells. We demonstrated that DDX11 interacts with Timeless, a component of the replication fork-protection complex, through a conserved peptide motif. The DDX11-Timeless interaction is critical for sister chromatid cohesion in interphase and mitosis. Immunofluorescence studies further revealed that cohesin association with chromatin requires DDX11. Finally, we demonstrated that DDX11 localises at nascent DNA by SIRF analysis. Moreover, we found that DDX11 promotes cohesin binding to the DNA replication forks in concert with Timeless and that recombinant purified cohesin interacts with DDX11 in vitro. Collectively, our results establish a critical role for the DDX11-Timeless interaction in coordinating DNA replication with sister chromatid cohesion, and have important implications for understanding the molecular basis of WABS.

We present evidence that Tim establishes a physical and functional interaction with DDX11, a super-family 2 iron-sulfur cluster DNA helicase genetically linked to the chromosomal instability disorder Warsaw breakage syndrome. Tim stimulates DDX11 unwinding activity on forked DNA substrates up to 10-fold and on bimolecular anti-parallel G-quadruplex DNA structures and three-stranded D-loop approximately 4–5-fold. Electrophoretic mobility shift assays revealed that Tim enhances DDX11 binding to DNA, suggesting that the observed stimulation derives from an improved ability of DDX11 to interact with the nucleic acid substrate. Surface plasmon resonance measurements indicate that DDX11 directly interacts with Tim. DNA fiber track assays with HeLa cells exposed to hydroxyurea demonstrated that Tim or DDX11 depletion significantly reduced replication fork progression compared to control cells; whereas no additive effect was observed by co-depletion of both proteins. Moreover, Tim and DDX11 are epistatic in promoting efficient resumption of stalled DNA replication forks in hydroxyurea-treated cells. This is consistent with the finding that association of the two endogenous proteins in the cell extract chromatin fraction is considerably increased following hydroxyurea exposure. Overall, our studies provide evidence that Tim and DDX11 physically and functionally interact and act in concert to preserve replication fork progression in perturbed conditions.