Yeast resolving enzyme CCE1 makes sequential cleavages in DNA junctions within the lifetime of the complex

CCE1 is a DNA junction-resolving enzyme of Saccharomyces cerevisiae. Such enzymes are required to make two symmetrically paired cleavages in order to resolve the four-way junction productively. Using a cruciform assay, we show here that CCE1 introduces two unilateral cleavages in a sequential manner. This requires that the protein remains bound to the junction, preventing branch migration of the point of strand exchange. From a detailed kinetic analysis, we find that the CCE1 cleavage at a given site is accelerated by a factor of 5-10 when it occurs subsequently to the initial cleavage. These properties ensure a productive resolution of the four-way junction and may be general for junction-resolving enzymes.     

Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS)

Deacetoxycephalosporin C synthase (DAOCS) is an iron(II) and 2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N to deacetoxycephalosporin C, the committed step in the biosynthesis of cephalosporin antibiotics. The crystal structure of DAOCS revealed that the C terminus of one molecule is inserted into the active site of its neighbor in a cyclical fashion within a trimeric unit. This arrangement has hindered the generation of crystalline enzyme-substrate complexes. Therefore, we constructed a series of DAOCS mutants with modified C termini. Oxidation of 2-oxoglutarate was significantly uncoupled from oxidation of the penicillin substrate in certain truncated mutants. The extent of uncoupling varied with the number of residues deleted and the penicillin substrate used. Crystal structures were determined for the DeltaR306 mutant complexed with iron(II) and 2-oxoglutarate (to 2.10 A) and the DeltaR306A mutant complexed with iron(II), succinate and unhydrated carbon dioxide (to 1.96 A). The latter may mimic a product complex, and supports proposals for a metal-bound CO(2) intermediate during catalysis.     

Alpha subunit variants of the human glycine receptor: primary structures, functional expression and chromosomal localization of the corresponding genes.

Two cDNAs encoding variants (alpha 1 and alpha 2) of the strychnine binding subunit of the inhibitory glycine receptor (GlyR) were isolated from a human fetal brain cDNA library. The predicted amino acid sequences exhibit approximately 99% and approximately 76% identity to the previously characterized rat 48 kd polypeptide. Heterologous expression of the human alpha 1 and alpha 2 subunits in Xenopus oocytes resulted in the formation of glycine-gated strychnine-sensitive chloride channels, indicating that both polypeptides can form functional GlyRs. Using a panel of rodent-human hybrid cell lines, the gene encoding alpha 2 was mapped to the short arm (Xp21.2-p22.1) of the human X chromosome. In contrast, the alpha 1 subunit gene is autosomally located. These data indicate molecular heterogeneity of the human GlyR at the level of alpha subunit genes.     

PHYTOPLANKTON PIGMENTS IN COASTAL LAKE MICHIGAN: DISTRIBUTIONS DURING THE SPRING ISOTHERMAL PERIOD AND RELATION WITH EPISODIC SEDIMENT RESUSPENSION1

Phytoplankton pigment distributions during the spring isothermal periods of 1998 and 1999 and their association with episodic sediment resuspension were characterized in coastal waters of southern Lake Michigan. Total and phylogenetic group chl a concentrations (derived using chemical taxonomy matrix factorization of diagnostic carotenoids) corresponded with assemblage and group biovolumes estimated from microscopic enumeration (P≤ 0.001). Diatoms and cryptophytes dominated assemblages and together typically comprised greater than 85% of relative chl a. Total chl a concentrations and both fucoxanthin·chl a ^{? 1} and alloxanthin·chl a ^{? 1} ratios were similar across depths (P> 0.05), indicating uniform distributions of and photophysiological states for assemblages and diatoms and cryptophytes, respectively, throughout the mixed water column. Total chl a concentrations were not always spatially uniform from near‐shore to offshore waters, with the greatest variability reflecting the influence of tributary inflows upon coastal assemblages. Sediment resuspension strongly influenced water column particle density and light climate; however, total and group chl a concentrations did not correspond with coefficients of K_d and suspended particulate matter concentrations (P> 0.05). The correspondence of both light attenuation and suspended particulate matter concentration with relative diatom chl a (P≤ 0.001) indicated an apparent association between sediment resuspension and diatoms. This, and the negative association (P≤ 0.0001) between relative diatom and cryptophyte chl a, corresponded with the spatial dominance of diatom and cryptophyte chl a in near‐shore and offshore waters, respectively. The presence of viable chl a and fucoxanthin within the surficial sediment layer, established this layer as a potential source of meroplanktonic diatoms for near‐shore assemblages.     

Crystal structure and biochemical analysis of the MutS.ADP.beryllium fluoride complex suggests a conserved mechanism for ATP interactions in mismatch repair

During mismatch repair ATP binding and hydrolysis activities by the MutS family proteins are important for both mismatch recognition and for transducing mismatch recognition signals to downstream repair factors. Despite intensive efforts, a MutS.ATP.DNA complex has eluded crystallographic analysis. Searching for ATP analogs that strongly bound to Thermus aquaticus (Taq) MutS, we found that ADP.beryllium fluoride (ABF), acted as a strong inhibitor of several MutS family ATPases. Furthermore, ABF promoted the formation of a ternary complex containing the Saccharomyces cerevisiae MSH2.MSH6 and MLH1.PMS1 proteins bound to mismatch DNA but did not promote dissociation of MSH2.MSH6 from mismatch DNA. Crystallographic analysis of the Taq MutS.DNA.ABF complex indicated that although this complex was very similar to that of MutS.DNA.ADP, both ADP.Mg(2+) moieties in the MutS. DNA.ADP structure were replaced by ABF. Furthermore, a disordered region near the ATP-binding pocket in the MutS B subunit became traceable, whereas the equivalent region in the A subunit that interacts with the mismatched nucleotide remained disordered. Finally, the DNA binding domains of MutS together with the mismatched DNA were shifted upon binding of ABF. We hypothesize that the presence of ABF is communicated between the two MutS subunits through the contact between the ordered loop and Domain III in addition to the intra-subunit helical lever arm that links the ATPase and DNA binding domains.     

Biochemical and structural characterisation of dehydroquinate synthase from the New Zealand kiwifruit Actinidia chinensis

One of the novel aspects of kiwifruit is the presence of a high level of quinic acid which contributes to the flavour of the fruit. Quinic acid metabolism intersects with the shikimate pathway, which is responsible for the de novo biosynthesis of primary and secondary aromatic metabolites. The gene encoding the enzyme which catalyses the second step of the shikimate pathway, dehydroquinate synthase (DHQS), from the New Zealand kiwifruit Actinidia chinensis was identified, cloned and expressed. A. chinensis DHQS was activated by divalent metal ions, and was found to require NAD⁺ for catalysis. The protein was crystallised and the structure was solved, revealing a homodimeric protein. Each monomer has a NAD⁺ binding site nestled between the distinct N- and C-terminal domains. In contrast to other microbial DHQSs, which show an open conformation in the absence of active site ligands, A. chinensis DHQS adopts a closed conformation. This is the first report of the structure of a DHQS from a plant source.     

Interactions Between Nile Perch, Lates niloticus, and Other Fishes in Lake Nabugabo, Uganda

The introduction of the predatory Nile perch, Lates niloticus, into the Lake Victoria basin coincided with a dramatic decline in fish species richness and diversity. This study focused on interactions between Nile perch and indigenous fishes in Lake Nabugabo, Uganda, a small satellite lake of Lake Victoria. We evaluated how the foraging impact of juvenile Nile perch on prey fishes varied with the size of the predator. We also evaluated the role of wetland ecotones in minimizing interaction between Nile perch and indigenous fishes. Wetland ecotones in Lake Nabugabo were characterized by complex structure (e.g., dense vegetation) and lower dissolved oxygen levels than non-wetland (exposed) areas. Nile perch (8.6–42.2cm, TL) were 3.7 times more abundant in offshore exposed areas than in inshore areas near wetland ecotones, and the proportion of Nile perch using wetland and exposed areas was independent of their body size. However, species richness was higher in waters at wetland ecotones than in exposed areas. Nile perch (5–35cm, TL) exhibited a shift in diet at approximately 30cm TL from feeding primarily on invertebrates to piscivory. Although the shift to piscivory occurred at approximately the same body size for Nile perch from both wetland and exposed habitats, the shift to piscivory was less abrupt in Nile perch captured near wetland ecotones. Nile perch from wetland areas consumed a greater diversity and a larger percentage of fish prey than those from exposed sites. However, the low abundance of Nile perch in wetland ecotones suggested that interaction between predator and prey in these areas is much reduced.     

Altered motor activity, exploration and anxiety in heterozygous neuregulin 1 mutant mice: implications for understanding schizophrenia

Human genetic studies have shown that neuregulin 1 (NRG1) is a potential susceptibility gene for schizophrenia. Nrg1 influences various neurodevelopmental processes, which are potentially related to schizophrenia. The neurodevelopmental theory of schizophrenia suggests that interactions between genetic and environmental factors are responsible for biochemical alterations leading to schizophrenia. To investigate these interactions and to match experimental design with the pathophysiology of schizophrenia, we applied a comprehensive behavioural phenotyping strategy for motor activity, exploration and anxiety in a heterozygous Nrg1 transmembrane domain mutant mouse model (Nrg1 HET) using different housing conditions and age groups. We observed a locomotion- and exploration-related hyperactive phenotype in Nrg1 HETs. Increased age had a locomotion- and exploration-inhibiting effect, which was significantly attenuated in mutant mice. Environmental enrichment (EE) had a stimulating influence on locomotion and exploration. The impact of EE was more pronounced in Nrg1 hypomorphs. Our study also showed a moderate task-specific anxiolytic-like phenotype for Nrg1 HETs, which was influenced by external factors. The behavioural phenotype detected in heterozygous Nrg1 mutant mice is not specific to schizophrenia per se, but the increased sensitivity of mutant mice to exogenous factors is consistent with the pathophysiology of schizophrenia and the neurodevelopmental theory. Our findings reinforce the importance of carefully controlling experimental designs for external factors and of comprehensive, integrative phenotyping strategies. Thus, Nrg1 HETs may, in combination with other genetic and drug models, help to clarify pathophysiological mechanisms behind schizophrenia.     

Mechanical anisotropy of ankyrin repeats

Red blood cells are frequently deformed and their cytoskeletal proteins such as spectrin and ankyrin-R are repeatedly subjected to mechanical forces. While the mechanics of spectrin was thoroughly investigated in vitro and in vivo, little is known about the mechanical behavior of ankyrin-R. In this study, we combine coarse-grained steered molecular dynamics simulations and atomic force spectroscopy to examine the mechanical response of ankyrin repeats (ARs) in a model synthetic AR protein NI6C, and in the D34 fragment of native ankyrin-R when these proteins are subjected to various stretching geometry conditions. Our steered molecular dynamics results, supported by AFM measurements, reveal an unusual mechanical anisotropy of ARs: their mechanical stability is greater when their unfolding is forced to propagate from the N-terminus toward the C-terminus (repeats unfold at ~60 pN), as compared to the unfolding in the opposite direction (unfolding force ～ 30 pN). This anisotropy is also reflected in the complex refolding behavior of ARs. The origin of this unfolding and refolding anisotropy is in the various numbers of native contacts that are broken and formed at the interfaces between neighboring repeats depending on the unfolding/refolding propagation directions. Finally, we discuss how these complex mechanical properties of ARs in D34 may affect its behavior in vivo.