Nuclease studies

In vivo studies of transcription on nucleosomes 2.1. Nuclease studies... 

Within this common framework individual nucleases studied to date differ greatly in their choice of catalytic residues, metal requirements and in their geometry of interaction with substrate. Detailed knowledge of the active site geometry of nucleases is usually very helpful for the most complete understanding of their mechanism, and this is most often accomplished using x-ray crystallographic methods. 

A. Paliwal, D. Asthagiri, D. P. Bossev, E. Paulaitis, Pressure denaturation of staphylococcal nuclease studied by neutron small-angle scattering and molecular simulation, Biophys. J. 87 (2004) 3479-3492. 

Modification of the Phosphodiester Backbone. Oligonucleotides having modified phosphate backbones have been extensively studied (46). Because altering the backbone makes derivatives generally more resistant to degradation by cellular nucleases, these materials have the potential to be more resilient antisense dmgs. 

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

Tucker, P.W., Hazen, E.E., Colton, F.A. Staphylococcal nuclease reviewed a prototypic study in contemporary enzymology. III. Correlation of fhe three-dimensional structure with the mechanisms of enzymatic action. Mol. Cell. Biochem. 

Kleimnan et al. 2008). In addition, synthetic siRNAs are also subject to degradation in vivo by nuclease activity. Besides side effects and instability, the efficient and specific delivery of the RNAi indncers to the target cell still requires optimization. Here we snmmarize the cnrrent statns of nncleic acid-based antiviral therapentics. The focns will be on antiviral strategies nsing antisense and RNAi technology. Additionally, antiviral ribozymes and aptamers will be discussed briefly, with a focus on recent studies. Gene therapy approaches and delivery systems are the subject of Chapter 11 of this book. 

Recently the use of another bifunctional reagent, glutaraldehyde, has been described for the stabilization of DNA complexes with cationic peptide CWK18 [104]. The authors of this paper, however, limited the study to the protective effects toward nuclease degradation. 

SI nuclease j Degrades single-stranded DNA. Removal of "hairpin" in synthesis of cDNA RNA mapping studies (both 5 and 3 ends). 

B. Solvolysis of Phosphoric Acid Derivatives.—Interest continues in neighbouring-group participation in the solvolysis of phosphate esters. As a potential model compound for investigating the mechanism of ribo-nuclease action, the phenyl hydrogen phosphate ester of c/j-3,4-tetrahydro-furandiol (24) has been the subject of a detailed study. Above (and probably also below) pH 4 hydrolysis gives solely the cyclic phosphate (25)... 

As already mentioned, Lippard and co-workers have also studied the -hydroxy dizinc unit found in metallohydrolases in a model system [Zn2L(/x-OI I)(/x-02PPh2)]2+, L = 2,7-bis [2-(2-pyridylethyl)-aminomethyl]-l,8-naphthyridine) 454 Hydrolysis of phosphodiesters and beta-lactams was studied and related to the PI nuclease and beta-lactamase enzymes. The dinuclear complex... 

Privalov et al (1989) studied the unfolded forms of several globular proteins [ribonuclease A, hen egg white lysozyme, apomyoglobin (apoMb), cytochrome c, and staphylococcal nuclease]. Unfolding was induced by 6 M Gdm-HCl at 10°C, heating to 80°C, or by low pH at 10°C with cross-links cleaved (reduction and carboxamidomethylation or removal of heme). The unfolded forms showed CD spectra (Fig. 27)... 

Administration is by direct injection of 0.05 ml product into the eye (intravitreal injection), initially once every 2 weeks and subsequently once every 4 weeks. Animal studies (rabbits) indicated that the product is cleared from the eye over the course of 7-10 days, with direct nuclease-mediated metabolism representing the primary route of elimination. The most commonly observed side effect is ocular inflammation, which typically occurs in one in every four patients. 

Deoxyribonucleic acid footprinting studies have shown that HMG domains A and B inhibit cleavage by nucleases over a 12- to 15-base-pair region centered around the platinum adduct (81). The HMG proteins can modulate cisplatin cytotoxicity by inhibition of the excinuclease-mediated removal of Pt-d(GpG) adducts from DNA (82). However, this hypothesis has been questioned because there is no evidence for cellular protein shielding of Pt-d(GpG) adducts from repair enzymes (83). 

Zhang Y, Wang Y, Liu B (2009) Peptide-mediated energy transfer between an anionic water-soluble conjugated polymer and Texas red labeled DNA for protease and nuclease activity study. Anal Chem 81 3731-3737... 

An in-depth study of DNA repair systems (Aravind et al., 1999a) has concluded that few, if any, repair proteins occur with identical collinear domain arrangements in all three kingdoms of life. Approximately 10 enzyme families of adenosine triphosphatases (ATPases), photolyases, helicases, and nucleases were identified that are all likely to have been present in the cenancestor. These enzymatic domains are accompanied in DNA repair proteins by numerous regulatory domains. This indicates that the domain architectures of these proteins are labile, with incremental addition and/or subtraction of domains to conserved cores to be a common phenomenon except in the most closely related species. 

The localization of bNOS to the human genome was accomplished by Kishimoto and coworkers70. These investigators used a rat cerebellar cDNA to obtain a human cDNA from Clontech. This cDNA was hybridized to Southern blots containing DNA from a battery of human-rodent somatic cell DNA. Since the blots had been shown to be selective, the authors showed that the cDNA hybridized to chromosome 12. By using restriction nucleases EcoRI and Hind in the assignment was made to 12 ql4-qter. One or two copies were indicated in vivo but reducing the hybrid conditions showed more bands. It is necessary to conduct further studies to see whether the other cDNA are derived from this or another clone. 

Similar results were obtained from reconstitution experiments with DNA and a non-cross-linked octamer (Thomas and Butler, 1978). Nucleosome-like particles were observed in the EM and a pattern of histone cross-linking comparable to that of native chromatin was obtained. However, only 140-base-pair repeats were obtained upon micrococcal nuclease digestion instead of 200-base-pair repeats obtained for native rat liver chromatin (Noll and Komberg, 1977). This indicates that, in the absence of HI, only core particles can be reconstituted. Nevertheless, these studies with both cross-linked and reassembled un-cross-linked histones demonstrate that the octamer is a complete biological functional unit retaining the information for folding the DNA around the histone core. 

Studies of proteolytic fragments of staphylococcal nuclease (Tan-iuchi and Anfinsen, 1969) and RNase A (Taniuchi, 1970) seemed to support this view. Taniuchi (1970), in summary remarks, said Thus, the minimum information of the specific folding of a protein requiring almost the entire amino acid sequence is observed with both staph-yloccocal nuclease and bovine pancreatic ribonuclease. ... 

In a further study, Taniuchi et al. (1977) have shown that in the association of overlapping fragments of staphylococcal nuclease, two different species of active enzyme are formed. On the basis of the products of limited proteolysis, structures for the two species were deduced. In one case a structure is proposed in which fragment 1-126 assumes native-like structure over the sequence 1-48, and all of fragment 50-149 assumes native-like structure. In the other case the structure is one in which fragment 1-126 assumes native-like structure over the sequence 1-110, while that part of fragment 50-149 in the sequence interval 111-149 assumes native-like structure. The interest of these results is enhanced by the finding that the two active species initially form in relative concentrations substantially different from their equilibrium concentrations. Thus, both a mobile equilibrium and substantial kinetic control of the early products are evident. Taniuchi et al. did not reach a clear-cut mechanistic conclusion from their studies. 

---