Structure of an XPF endonuclease with and without DNA suggests a model for substrate recognition

The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH)(2) domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3' flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes.     

Both endogenous and environmental factors affect embryo proliferation in the polyembryonic wasp Copidosoma floridanum

Copidosoma floridanum is a polyembryonic, parasitic wasp of the moth Trichoplusia ni. Following oviposition into a host, the C. floridanum egg initially undergoes complete (holoblastic) cleavage to form a single morula stage embryo. This embryo then undergoes a proliferation phase in which multiple, secondary morulae develop. C. floridanum has also evolved a caste system whereby some secondary morulae develop into soldier larvae whose function is defense whereas others develop into reproductive larvae that become adult wasps. In the current study, we conducted manipulative and candidate gene studies to identify factors affecting the proliferation phase of C. floridanum development. Transplantation of morulae of different ages into different host stages indicated that both embryo age and host environment affected the total number of offspring produced per morula. Morula age and brood size also significantly affected whether offspring of one or both castes were produced in a brood. In contrast, the host environment did not significantly affect caste formation. A putative homolog of the gene hedgehog (Cf-hh) was partially cloned from C. floridanum. In situ hybridization studies indicated that Cf-hh was expressed in secondary morulae during the proliferation phase of development, suggesting a possible role for the Hh signaling pathway in the evolution of polyembryony.     

Genetic rescue of a Toxoplasma gondii conditional cell cycle mutant

Growth rate is a major pathogenesis factor in the parasite Toxoplasma gondii; however, how cell division is controlled in this protozoan is poorly understood. Herein, we show that centrosomal duplication is an indicator of S phase entry while centrosome migration marks mitotic entry. Using the pattern of centrosomal replication, we confirmed that mutant ts11C9 undergoes a bimodal cell cycle arrest that is characterized by two subpopulations containing either single or duplicated centrosomes which correlate with the bipartite genome distribution observed at the non-permissive temperature. Genetic rescue of ts11C9 was performed using a parental RH strain cDNA library, and the cDNA responsible for conferring temperature resistance (growth at 40 degrees C) was recovered by recombination cloning. A single T. gondii gene encoding the protein homologue of XPMC2 was responsible for genetic rescue of the temperature-sensitive defect in ts11C9 parasites. This protein is a known suppressor of mitotic defects, and in tachyzoites, TgXPMC2-YFP localized to the parasite nucleus and nucleolus which is consistent with the expected subcellular localization of critical mitotic factors. Altogether, these results demonstrate that ts11C9 is a conditional mitotic mutant containing a single defect which influences two distinct control points in the T. gondii tachyzoite cell cycle.     

The structural definition of adducts of stoichiometry MX:dppx (1:1) M = Cu, Ag, X = simple anion, dppx=Ph2P(CH2)xPPh2, x = 3-6

Single crystal X-ray structural characterizations are recorded for a wide range of adducts of the form MX:dppx (1:1)_(n), M = silver(I) (predominantly), copper(I), X = simple (pseudo-) halide or oxy-anion (the latter spanning, where accessible, perchlorate, nitrate, carboxylate - a range of increasing basicity), dppx=bis(diphenylphosphino)alkane, Ph₂P(CH₂)_xPPh₂, x = 3-6. Adducts are defined of two binuclear forms: (i) [LM(μ-X)₂L], with each ligand chelating a single metal atom, and (ii) [M(μ-X)₂(μ-(P-L-P′))₂M′] where both ligands L and halides bridge the two metal atoms; a few adducts are defined as polymers, the ligands connecting M(μ-X)₂M′ kernels, this motif persisting in all forms. Synthetic procedures for all adducts have been reported. All compounds have been characterized both in solution (¹H, ¹³C, ³¹P NMR, ESI MS) and in the solid state (IR).     

Headloop suppression PCR and its application to selective amplification of methylated DNA sequences

Selective amplification in PCR is principally determined by the sequence of the primers and the temperature of the annealing step. We have developed a new PCR technique for distinguishing related sequences in which additional selectivity is dependent on sequences within the amplicon. A 5′ extension is included in one (or both) primer(s) that corresponds to sequences within one of the related amplicons. After copying and incorporation into the PCR product this sequence is then able to loop back, anneal to the internal sequences and prime to form a hairpin structure—this structure is then refractory to further amplification. Thus, amplification of sequences containing a perfect match to the 5′ extension is suppressed while amplification of sequences containing mismatches or lacking the sequence is unaffected. We have applied Headloop PCR to DNA that had been bisulphite-treated for the selective amplification of methylated sequences of the human GSTP1 gene in the presence of up to a 10⁵-fold excess of unmethylated sequences. Headloop PCR has a potential for clinical application in the detection of differently methylated DNAs following bisulphite treatment as well as for selective amplification of sequence variants or mutants in the presence of an excess of closely related DNA sequences.     

Thioredoxin and dihydrolipoic acid inhibit elastase activity in cystic fibrosis sputum

Excessive neutrophil elastase activity within airways of cystic fibrosis (CF) patients results in progressive lung damage. Disruption of disulfide bonds on elastase by reducing agents may modify its enzymatic activity. Three naturally occurring dithiol reducing systems were examined for their effects on elastase activity: 1) Escherichia coli thioredoxin (Trx) system, 2) recombinant human thioredoxin (rhTrx) system, and 3) dihydrolipoic acid (DHLA). The Trx systems consisted of Trx, Trx reductase, and NADPH. As shown by spectrophotometric assay of elastase activity, the two Trx systems and DHLA inhibited purified human neutrophil elastase as well as the elastolytic activity present in the soluble phase (sol) of CF sputum. Removal of any of the three Trx system constituents prevented inhibition. Compared with the monothiols N-acetylcysteine and reduced glutathione, the dithiols displayed greater elastase inhibition. To streamline Trx as an investigational tool, a stable reduced form of rhTrx was synthesized and used as a single component. Reduced rhTrx inhibited purified elastase and CF sputum sol elastase without NADPH or Trx reductase. Because Trx and DHLA have mucolytic effects, we investigated changes in elastase activity after mucolytic treatment. Unprocessed CF sputum was directly treated with reduced rhTrx, the Trx system, DHLA, or DNase. The Trx system and DHLA did not increase elastase activity, whereas reduced rhTrx treatment increased sol elastase activity by 60%. By contrast, the elastase activity after DNase treatment increased by 190%. The ability of Trx and DHLA to limit elastase activity combined with their mucolytic effects makes these compounds potential therapies for CF.     

Crystal structures of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase-GMP and -IMP complexes: comparison of purine binding interactions with the XMP complex.

The crystal structures of the guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP) complexes of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT) have been determined at 1.65 and 1.90 A resolution. These complexes, which crystallize in space groups P2(1) (a = 65.45 A, b = 90.84 A, c = 80. 26 A, and beta = 92.53 degrees ) and P2(1)2(1)2(1) (a = 84.54 A, b = 102.44 A, and c = 108.83 A), each comprise a tetramer in the crystallographic asymmetric unit. All active sites in the tetramers are fully occupied by the nucleotide. Comparison of these structures with that of the xanthosine 5'-monophosphate (XMP)-pyrophosphate-Mg(2+) ternary complex reported in the following article [Héroux, A., et al. (1999) Biochemistry 38, 14495-14506] shows how T. gondii HGPRT is able to recognize guanine, hypoxanthine, and xanthine as substrates, and suggests why the human enzyme cannot use xanthine efficiently. Comparison with the apoenzyme reveals the structural changes that occur upon binding of purines and ribose 5'-phosphate to HGPRT. Two structural features important to the HGPRT mechanism, a previously unrecognized active site loop (loop III', residues 180-184) and an active site peptide bond (Leu78-Lys79) that adopts both the cis and the trans configurations, are presented.     

The dynamic structure underlying subthreshold oscillatory activity and the onset of spikes in a model of medial entorhinal cortex stellate cells

Medial entorhinal cortex layer II stellate cells display subthreshold oscillations (STOs). We study a single compartment biophysical model of such cells which qualitatively reproduces these STOs. We argue that in the subthreshold interval (STI) the seven-dimensional model can be reduced to a three-dimensional system of equations with well differentiated times scales. Using dynamical systems arguments we provide a mechanism for generations of STOs. This mechanism is based on the “canard structure,” in which relevant trajectories stay close to repelling manifolds for a significant interval of time. We also show that the transition from subthreshold oscillatory activity to spiking (“canard explosion”) is controlled in the STI by the same structure. A similar mechanism is invoked to explain why noise increases the robustness of the STO regime. Taking advantage of the reduction of the dimensionality of the full stellate cell system, we propose a nonlinear artificially spiking (NAS) model in which the STI reduced system is supplemented with a threshold for spiking and a reset voltage. We show that the synchronization properties in networks made up of the NAS cells are similar to those of networks using the full stellate cell models. In memory of Angel A. Alonso