Proliferation Index: A Continuous Model to Predict Prognosis in Patients with Tumours of the Ewing's Sarcoma Family

The prognostic value of proliferation index (PI) and apoptotic index (AI), caspase-8, -9 and -10 expression have been investigated in primary Ewing's sarcoma family of tumours (ESFT). Proliferating cells, detected by immunohistochemistry for Ki-67, were identified in 91% (91/100) of tumours with a median PI of 14 (range 0–87). Apoptotic cells, identified using the TUNEL assay, were detected in 96% (76/79) of ESFT; the median AI was 3 (range 0–33). Caspase-8 protein expression was negative (0) in 14% (11/79), low (1) in 33% (26/79), medium (2) in 38% (30/79) and high (3) in 15% (12/79) of tumours, caspase-9 expression was low (1) in 66% (39/59) and high (3) in 34% (20/59), and caspase-10 protein was low (1) in 37% (23/62) and negative (0) in 63% (39/62) of primary ESFT. There was no apparent relationship between caspase-8, -9 and -10 expression, PI and AI. PI was predictive of relapse-free survival (RFS; p = 0.011) and overall survival (OS; p = <0.001) in a continuous model, whereas AI did not predict outcome. Patients with tumours expressing low levels of caspase-9 protein had a trend towards a worse RFS than patients with tumours expressing higher levels of caspase-9 protein (p = 0.054, log rank test), although expression of caspases-8, -9 and/or -10 did not significantly predict RFS or OS. In a multivariate analysis model that included tumour site, tumour volume, the presence of metastatic disease at diagnosis, PI and AI, PI independently predicts OS (p = 0.003). Consistent with previous publications, patients with pelvic tumours had a significantly worse OS than patients with tumours at other sites (p = 0.028); patients with a pelvic tumour and a PI≥20 had a 6 fold-increased risk of death. These studies advocate the evaluation of PI in a risk model of outcome for patients with ESFT

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genome

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes

Mechanism of DNA cleavage by the DNA/RNA-non-specific Anabaena sp. PCC 7120 endonuclease NucA and its inhibition by NuiA

A structural model of the DNA/RNA non-specific endonuclease NucA from Anabaena sp. PCC7120 that has been obtained on the basis of the three-dimensional structure of the related Serratia nuclease, suggests that the overall architecture of the active site including amino acid residues H124, N155 and E163 (corresponding to H89, N119 and E127 in Serratia nuclease) is similar in both nucleases. Substitution of these residues by alanine leads to a large reduction in activity (<0.1%), similarly as observed for Serratia nuclease demonstrating that both enzymes share a similar mechanism of catalysis with differences only in detail. NucA is inhibited by its specific polypeptide inhibitor with a K1 value in the subpicomolar range, while the related Serratia nuclease at nanomolar concentrations is only inhibited at an approximately 1000-fold molar excess of NuiA. The artificial chromophoric substrate deoxythymidine 3',5'-bis-(p-nitrophenyl phosphate) is cleaved by NucA as well as by Serratia nuclease. Cleavage of this analogue by NucA, however, is not inhibited by NuiA, suggesting that small molecules gain access to the active site of NucA in the enzyme-inhibitor complex under conditions where cleavage of DNA substrates is completely inhibited. The active site residue E163 seems to be the main target amino acid for inhibition of NucA by NuiA, but R93, R122 and R167 (corresponding to K55, R87, R131 in Serratia nuclease) are also involved in the NucA/NuiA interaction. NuiA deletion mutants show that the structural integrity of the N and C-terminal region of the inhibitor is important for complex formation with NucA and inhibition of nuclease activity. Based on these results a mechanism of DNA cleavage by NucA and its inhibition by NuiA is proposed. (C) 2000 Academic Press

Identification of functionally relevant histidine residues in the apoptotic nuclease CAD

The caspase-activated DNase CAD (DFF40/CPAN) degrades chromosomal DNA during apoptosis. Chemical modification with DEPC inactivates the enzyme, suggesting that histidine residues play a decisive role in the catalytic mechanism of this nuclease. Sequence alignment of murine CAD with four homologous apoptotic nucleases reveals four completely (His242, His263, His304 and His308) and two partially (His127 and His313) conserved histidine residues in the catalytic domain of the enzyme. We have changed these residues to asparagine and characterised the variant enzymes with respect to their DNA cleavage activity, structural integrity and oligomeric state. All variants show a decrease in activity compared to the wild-type nuclease as measured by a plasmid DNA cleavage assay. H242N, H263N and H313N exhibit DNA cleavage activities below 5% and H308N displays a drastically altered DNA cleavage pattern compared to wild-type CAD. Whereas all variants but one have the same secondary structure composition and oligomeric state, H242N does not, suggesting that His242 has an important structural role. On the basis of these results, possible roles for His127, His263, His304, His308 and His313 in DNA binding and cleavage are discussed for murine CAD

Involvement of conserved histidine, lysine and tyrosine residues in the mechanism of DNA cleavage by the caspase-3 activated DNase CAD

The caspase-activated DNase (CAD) is involved in DNA degradation during apoptosis. Chemical modification of murine CAD with the lysine-specific reagent 2,4,6-trinitrobenzenesulphonic acid and the tyrosine-specific reagent N-acetylimidazole leads to inactivation of the nuclease, indicating that lysine and tyrosine residues are important for DNA cleavage by this enzyme. The presence of DNA or the inhibitor ICAD-L protects the enzyme from modification. Amino acid substitution in murine CAD of lysines and tyrosines conserved in CADs from five different species leads to variants with little if any catalytic activity, but unaltered DNA binding (K155Q, K301Q, K310Q, Y247F), with the exception of Y170F, which retains wild-type activity. Similarly, as observed for the previously characterised H242N, H263N, H308N and H313N variants, the newly introduced His→Asp/Glu or Arg exchanges lead to variants with <1% of wild-type activity, with two exceptions: H313R shows wild-type activity, and H308D at pH 5.0 exhibits ∼5% of wild-type activity at this pH. Y170F and H313R produce a specific pattern of fragments, different from wild-type CAD, which degrades DNA non-specifically. The recombinant nuclease variants produced in Escherichia coli were tested for their ability to form nucleolytically active oligomers. They did not show any significant deviation from the wild-type enzyme. Based on these and published data possible roles of the amino acid residues under investigation are discussed

Biochemical characterization of Anabaena sp. strain PCC 7120 non- specific nuclease NucA and its inhibitor NuiA

We have established overexpression systems and purification protocols for NucA and NuiA, a sugar non-specific nuclease and its protein inhibitor from Anabaena sp. strain PCC 7120, in order to characterize these proteins in detail. CD spectroscopy revealed that NucA has a similar secondary-structure composition, 13% a helix and 20% β sheet, to the related Serratia nuclease, while NuiA represents a protein with a higher α-helical (29%) and β-sheet (24%) content than NucA. Denaturation experiments showed that the stabilities of NucA and NuiA are in the typical range for proteins of mesophilic organisms, NuiA with ΔG(o)(H2O)= 63.4 J* mol-1, (residue), being slightly more stable than its target NucA with ΔΔ(o)(H2O)= 46.3 J * mol- 1(residue). The nuclease requires divalent metal ions as cofactors, the optimum concentration being around 5 mM for Mn2+ or Mg2+. The order of effectiveness of various divalent cations to function as cofactors for the hydrolytic activity of NucA is Mn2+ = Co2+ > Mg2+≤ Ni2+> Ca2+ = Cd2+ at a concentration of 5 mM. Nuclease activity decreases with increasing concentration of monovalent salt. The activity of NucA shows a pH optimum at pH 5.5-7.5. The temperature optimum is around 35°C, the activation energy was calculated to be 53 kJ mol-1. The specific activity of the nuclease towards high molecular-mass DNA is 8.4X 106 Kunitz-units * mg-1, which means that NucA is one of the most active nucleases known. Kinetic constants for the cleavage of various DNA and RNA substrates by NucA are all in the range K(m) ≤ 0.1 mg * ml- and k(cm) 1000 s-1. As other non-specific nucleases, NucA exhibits sequence preferences, similar to the related Serratia nuclease, NucA avoids cleavage of d(A) d(T) tracts. The nucleolytic activity of NucA is completely inhibited at equimolar concentrations of nuclease and inhibitor. An ultracentrifugation analysis showed that NucA and NuiA form a 1:1 complex. The interaction of NucA with NuiA was also investigated by CD spectroscopy and revealed no major conformational changes upon complex formation of the two proteins

Chemical Rescue of Active Site Mutants of S. pneumoniae Surface Endonuclease EndA and Other Nucleases of the HNH Family by Imidazole

The His-Asn-His (HNH) motif characterizes the active sites of a large number of different nucleases such as homing endonucleases, restriction endonucleases, structure-specific nucleases and, in particular, nonspecific nucleases. Several biochemical studies have revealed an essential catalytic function for the first amino acid of this motif in HNH nucleases. This histidine residue was identified as the general base that activates a water molecule for a nucleophilic attack on the sugar phosphate backbone of nucleic acids. Replacement of histidine by an amino acid such as glycine or alanine, which lack the catalytically active imidazole side chain, leads to decreases of several orders of magnitude in the nucleolytic activities of members of this nuclease family. We were able, however, to restore the activity of HNH nuclease variants (i.e., EndA (Streptococcus pneumoniae), SmaNuc (Serratia marcescens) and NucA (Anabaena sp.)) that had been inactivated by His→Gly or His→Ala substitution by adding excess imidazole to the inactive enzymes in vitro. Imidazole clearly replaces the missing histidine side chain and thereby restores nucleolytic activity. Significantly, this chemical rescue could also be observed in vivo (Escherichia coli). The in vivo assay might be a promising starting point for the development of a high-throughput screening system for functional EndA inhibitors because, unlike the wild-type enzyme, the H160G and H160A variants of EndA can easily be produced in E. coli. A simple viability assay would allow inhibitors of EndA to be identified because these would counteract the toxicities of the chemically rescued EndA variants. Such inhibitors could be used to block the nucleolytic activity of EndA, which as a surface-exposed enzyme in its natural host destroys the DNA scaffolds of neutrophil extracellular traps (NETs) and thereby allows S. pneumoniae to escape the innate immune response. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Genetic engineering of Escherichia coli to produce a 1:1 complex of the Anabaena sp. PCC 7120 nuclease NucA and its inhibitor NuiA

A series of T7-promoter based bicistronic expression vectors was constructed in order to produce the complex of the Anabaena sp. PCC 7120 DNA/RNA non-specific nuclease NucA and its inhibitor NuiA. With all constructs, tandem expression of nucA and nuiA results in aggregation and inclusion body formation of NucA, independent of the order of the genes, the relative expression of the two proteins and the temperature applied during expression. Two constructs in which nuiA is the first and nucA the second cistron lead to an approximately one order of magnitude higher expression of nuiA compared with nucA. In these cells inclusion bodies are formed which contain NucA and NuiA in a 1:1 molar ratio. The complex can be solubilized with 6 M urea after disruption of the cells by sonication, renatured by dialysis and purified to homogeneity. 2 mg of the complex are obtained from 1:1 Escherichia coli culture. As shown by gel filtration and analytical ultracentrifugation, our system leads to a highly pure and homogeneous complex preparation, as required for biophysical and structural studies. Thus, our new method is a superior alternative for the production of the NucA/NuiA complex in which separately produced nuclease and inhibitor are mixed, and an excess of one or the other component, as well as aggregates of NucA, have to be removed from the preparation. (C) 2000 Elsevier Science B.V