Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.31.1 (micrococcal nuclease)
2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

By a combination of NMR docking and model building, the substrate binding site on staphylococcal nuclease was found to accommodate a trinucleotide and to consist of three subsites, each interacting with a single nucleotidyl unit of DNA. Binding of the essential Ca2+ activator and substrate cleavage occur between subsites 1 and 2. Hence, catalytically productive binding would span subsites 1 and 2 while nonproductive binding would span subsites 2 and 3. Lys-49 is near subsite 1, and Lys-84 and Tyr-115 interact with substrates at subsite 3 [Weber, D.J., Gittis, A.G., Mullen, G.P., Abeygunawardana, C., Lattman, E.E., Mildvan, A.S. Proteins 13:275-287, 1992]. The proposed locations of these subsites were independently tested by the effects of the K49A, K84A, and Y115A mutations of staphylococcal nuclease on the binding of Mn2+, Ca2+, and the dinucleotide and trinucleotide substrates, 5'-pdTdA, dTdA, and dTdAdG. These three mutants have previously been shown to be fully active and to have CD and 2D NMR spectra very similar to those of the wild-type enzyme (Chuang, W.-J., Weber, D.J., Gittis, A.G., Mildvan, A.S. Proteins 17:36-48, 1993). All three mutant enzymes and their pdTdA and dTdA complexes (but not their dTdAdG complex) bind Mn2+ and Ca2+ more weakly than the wild-type enzyme by factors ranging from 2 to 11. The presence of a terminal phosphate as in 5'-pdTdA raises the affinity of the substrate for staphylococcal nuclease and its three mutants by two orders of magnitude and for the corresponding enzyme-metal complexes by three to four orders of magnitude, suggesting that the terminal phosphate is coordinated by the enzyme-bound divalent cation. Such complexation would result in the nonproductive binding of 5'-pdTdA at subsites 2 and 3. Accordingly, the K84A and Y115A mutations significantly weaken the binding of 5'-pdTdA and its metal to staphylococcal nuclease by factors of 2.2 to 37.8, while the K49A mutation has much smaller or no effect. Such nonproductive binding explains the low activity of staphylococcal nuclease with small substrates, especially those with a terminal phosphate. Similarly, the K84A and Y115A mutations weaken the binding of dTdA and its metal complexes to the enzyme by factors of 3.4 to 13.1 while the K49A mutation has smaller effects indicating significant nonproductive binding of dTdA. The trinucleotide dTdAdG binds more tightly to wild-type and mutant staphylococcal nuclease and to its metal complexes than does the dinucleotide dTdA by factors of 2.4 to 12.2, reflecting the occupancy of an additional subsite.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Magnetic resonance studies of the binding of oligonucleotide substrates to mutants of staphylococcal nuclease. 814 23

The presence of bound substrate protein increases the thermostability of hsp70 molecular chaperones (heat shock proteins of molecular mass 70 kDa). Complexes between hsp70 and unfolded substrate proteins were isolated by size-exclusion high performance liquid chromatography. The isolated complexes were observed to dissociate at a significant rate even in the absence of ATP. The presence of ADP caused a substantial increase in the stability of the complex. Both ADP and inorganic phosphate were found to inhibit the ATP-induced dissociation of complex. ADP was also observed to increase both the rate of complex formation and its stability as a function of temperature, suggesting an important regulatory role for nucleotides during heat shock. Circular dichroism and fluorescence studies of the complex between DnaK and a thermally unstable mutant of staphylococcal nuclease indicate that the bound substrate protein is significantly unfolded. A model for hsp70 cycle of complex formation and dissociation, which accounts for the regulatory role of nucleotides, is proposed.
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PMID:hsp70-protein complexes. Complex stability and conformation of bound substrate protein. 817 36

Food-derived aminoimidazoazarenes have been shown to be mutagenic and carcinogenic and to form covalent DNA adducts. 32P-Post-labelling analysis of DNA modified with these heterocyclic amines (HA), including 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,4-dimethylimidazo [4,5-f]quinoline (MeIQ), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) has resulted in considerable interlaboratory variation in the characteristic patterns of DNA adduct spots, with up to six being detected for each compound. Similar complex patterns were observed when azido-derivatives of HA were photoreacted with calf thymus DNA. When deoxyguanosine 3'-monophosphate was modified with the azido derivatives and analysed using the 32P-post-labelling procedure, one major spot was observed for IQ, 4,8-DiMeIQx, 7,8-DiMeIQx or PhIP and two major spots for MeIQ or MeIQx. In each case, these adducts were chromatographically indistinguishable from the major adducts formed with DNA. No major adduct spots were observed when 3'-phosphate derivatives of deoxyadenosine, deoxycytidine or thymidine were reacted with the azido-derivatives of HA. In an attempt to identify the additional spots, azido derivatives of PhIP or IQ were reacted with the synthetic homopolymer poly(dG).poly(dC), the alternating copolymer poly(dC-dG) or a synthetic oligonucleotide (TTT-GTTTTTTCTTTCCCT): in each case a reduced number of adduct spots were detected. The introduction of an additional nuclease P1 hydrolysis step following the labelling reaction further reduced the number of adduct spots to only one or two major spots. Reversed-phase HPLC analysis showed that the number of peaks of radioactivity was also reduced to one or two, presumably corresponding to the [32P]-5'-monophosphate deoxyguanosine adducts. We suggest that many of the additional spots commonly observed in conventional 32P-post-labelling analysis of HA-modified DNA are adducted oligonucleotides that are partly resistant to hydrolysis by micrococcal nuclease and spleen phosphodiesterase but are susceptible to hydrolysis by nuclease P1.
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PMID:32P-post-labelling analysis of DNA adducts formed by food-derived heterocyclic amines: evidence for incomplete hydrolysis and a procedure for adduct pattern simplification. 820 90

The 32P-postlabelling methodology for analysis of DNA adducts derived from carcinogens containing one aromatic ring (e.g., safrole, styrene oxide, benzene metabolites, 1-nitrosoindole-3-acetonitrile) or a bulky non-aromatic moiety (e.g., mitomycin C, diaziquone) is reviewed. Six steps are involved: digestion of DNA to 3'-nucleotides, enrichment of adducts, 32P-labelling of adducts, separation of labelled adducts by TLC, detection, and quantitation. The first step, DNA digestion with micrococcal nuclease and spleen phosphodiesterase, is applicable to DNA modified with most carcinogens independent of their size and structure. Of the two commonly used procedures for enrichment of aromatic adducts in DNA digests, the nuclease P1 treatment is substantially more effective than butanol extraction for small aromatic and bulky non-aromatic adducts. For initial purification of these adducts from unadducted material after 32P-labelling, multi-directional polyethyleneimine (PEI)-cellulose TLC using 1 M sodium phosphate, pH 6.0, as the D1 solvent is not suitable, because they are not retained on PEI-cellulose under these conditions. A higher concentration of sodium phosphate (e.g., 2.3 M) or development with D1 and D3 solvents in the same direction helps to retain adducts of safrole and of benzene metabolites. Also, transfer of adducts from multiple cut-outs above the origin after D1 chromatography, as adopted for analysis of I-compounds, is potentially applicable. However, initial purification by reverse-phase TLC, followed by in situ transfer and resolution by PEI-cellulose TLC has been found to be most effective for these adducts. Reverse-phase TLC at 4 degrees C or in a stronger salt solution further improves retention of some adducts (e.g., mitomycin C and diaziquone adducts). For adduct separation by PEI-cellulose TLC, salt solutions with or without urea are used.
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PMID:32P-postlabelling analysis of small aromatic and of bulky non-aromatic DNA adducts. 822 92

In the X-ray structure of the ternary staphylococcal nuclease-Ca(2+)-3',5'-pdTp complex, the conformation of the bound inhibitor 3',5'-pdTp is distorted by Lys-70* and Lys-71* from an adjacent molecule of the enzyme in the crystal lattice (Loll, P. J. and Lattman, E. E. Proteins 5:183-201, 1989; Serpersu, E. H., Hibler, D. W., Gerlt, J. A., and Mildvan, A. S. Biochemistry 28:1539-1548, 1989). Since this interaction does not occur in solution, the NMR docking procedure has been used to correct this problem. Based on 8 Co(2+)-nucleus distances measured by paramagnetic effects on T1, and 9 measured and 45 lower limit interproton distances determined by 1D and 2D NOE studies of the ternary Ca2+ complex, the conformation of enzyme-bound 3',5'-pdTp is high-anti (chi = 58 +/- 10 degrees) with a C2' endo/O1' endo sugar pucker (delta = 143 +/- 2 degrees), (-) synclinal about the C3'-O3' bond (epsilon = 273 +/- 4 degrees), trans, gauche about the C4'-C5' bond (gamma = 301 +/- 29 degrees) and either (-) or (+) clinal about the C5'-O5' bond (beta = 92 +/- 8 degrees or 274 +/- 3 degrees). The structure of 3',5'-pdTp in the crystalline complex differs due to rotations about the C4'-C5' bond (gamma = 186 +/- 12 degrees, gauche, trans) and the C5'-O5' bond [beta = 136 +/- 10 degrees, (+) anticlinal]. The undistorted conformation of enzyme-bound metal-3',5'-pdTp determined by NMR was docked into the X-ray structure of the enzyme, using 19 intermolecular NOEs from ring proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the inhibitor. van der Waals overlaps were then removed by energy minimization. Subsequent molecular dynamics and energy minimization produced no significant changes, indicating the structure to be in a global rather than in a local minimum. While the metal-coordinated 5'-phosphate of the NMR-docked structure of 3',5'-pdTp overlaps with that in the X-ray structure, and similarly receives bifunctional hydrogen bonds from both Arg-35 and Arg-87, the thymine, deoxyribose, and 3'-phosphate are significantly displaced from their positions in the X-ray structure, with the 3'-phosphate receiving hydrogen bonds from Lys-49 rather than from Lys-84 and Tyr-85. The repositioned thymine ring permits hydrogen bonding to the phenolic hydroxyl of Tyr-115.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:NMR docking of the competitive inhibitor thymidine 3',5'-diphosphate into the X-ray structure of staphylococcal nuclease. 823 42

In the X-ray structure of the staphylococcal nuclease-Ca(2+)-3',5'-pdTp complex, the conformation of the inhibitor 3',5'-pdTp is distorted by Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5:183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3',5'-pdTp in the staphylococcal nuclease-metal-pdTp complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D.J., Serpersu, E.H., Gittis, A.G., Lattman, E.E., Mildvan, A.S. (preceding paper)]. In the NMR-docked structure, the 5'-phosphate of 3',5'-pdTp overlaps with that in the X-ray structure. However, the 3'-phosphate accepts a hydrogen bond from Lys-49 (2.89 A) rather than from Lys-84 (8.63 A), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 A) which does not occur in the X-ray structure (5.28 A). These interactions have been tested by binding studies of 3',5'-pdTp, Ca2+, and Mn2+ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca2+ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While the K84A mutation did not significantly weaken 3',5'-pdTp binding to the enzyme (1.5 +/- 0.7 fold), the K49A mutation weakened 3',5'-pdTp binding to the enzyme by the factor of 4.4 +/- 1.8-fold. Similarly, the Y115A mutation weakened 3',5'-pdTp binding to the enzyme 3.6 +/- 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca(2+)-3',5'-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3',5'-pdTp than by the X-ray structure.
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PMID:Mutational tests of the NMR-docked structure of the staphylococcal nuclease-metal-3',5'-pdTp complex. 823 43

The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serratia nuclease. Most of the natural substrates are cleaved with similar Kcat and K(m) values, the Kcat/K(m) ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA).poly(dT) or poly(dG).poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K(m), respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-Rp-oxygen of the phosphate group 3' adjacent to the scissile phosphodiester bond is essential for cleavage, as only the Rp-phosphorothioate supports cleavage at the 5' adjacent phosphodiester bond. Furthermore, the modified bond itself is only cleaved in the Rp-diastereomer, albeit 1000 times more slowly than the corresponding unmodified phosphodiester bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the phosphodiester bond that is the rate-limiting step of the reaction. Finally, it is shown that Serratia nuclease accepts thymidine 3',5'-bis(p-nitrophenyl)phosphate as a substrate and cleaves it at its 5'-end to produce nitrophenol and thymidine 3'-(p-nitrophenylphosphate) 5-phosphate. The rate of cleavage of this artificial substrate, however, is 6-7 orders of magnitude smaller than the rate of cleavage of macromolecular DNA or RNA.
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PMID:Kinetic analysis of the cleavage of natural and synthetic substrates by the Serratia nuclease. 891 58

Postlabeling can be one of the most sensitive methods for the measurement of DNA adducts. However, for the determination of alkylated adducts, essential requirements are standards which must be fully chemically characterized. In order to develop a postlabeling assay for monitoring exposure to genotoxic ethylating agents, the reaction of diethyl sulfate with 2'-deoxynucleoside 3'- and 5'-monophosphates was examined. The adducts generated were fully characterized using HPLC, electrospray tandem mass spectrometry, UV, and postlabeling. The major product was the phosphodiester derived from alkylation of the phosphate, and alkylation of the base occurred to a lesser extent. The phosphodiester standard, 2'-deoxyguanosine 3'-(mono-O-ethyl phosphate) (3'Et-pdG), was used to develop a postlabeling assay for the detection of this adduct in DNA samples. Since alkylated phosphodiesters in DNA are not susceptible to the actions of micrococcal nuclease and calf spleen phosphodiesterase, they can be obtained as alkylated phosphodiester dinucleosides from DNA. Nuclease P1 was used as enhancement step which allowed the separation of these adducted phosphotriesters from the unmodified nucleotides by HPLC. Subsequent hydrolysis of the phosphotriester dinucleosides in alkali yielded phosphodiesters, including 3'Et-pdG, which was efficiently postlabeled. This approach was shown to be capable of detecting this adduct in liver DNA from mice treated intraperitoneally with N-nitrosodiethylamine.
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PMID:Detection and characterization of two major ethylated deoxyguanosine adducts by high performance liquid chromatography, electrospray mass spectrometry, and 32P-postlabeling. Development of an approach for detection of phosphotriesters. 907 5

8-Oxo-2'-deoxyguanosine (8-oxo-dG) is emerging as a useful marker for oxidative DNA damage. Reported basal levels determined by 32P-postlabeling (PPL) method were 10-fold or more higher than those obtained with HPLC/electrochemical detection (ECD). This discrepancy was investigated. In commercial calf thymus DNA, levels of 4 +/- 1 and 64 +/- 14 8-oxo-dG per 10(6) 2'-deoxynucleosides (dN) were measured by the standard HPLC/ECD and PPL methods, respectively. DNA digestion by micrococcal nuclease/spleen phosphodiesterase and nuclease P1 (as used in the standard PPL method), followed by ECD analysis resulted in a level of 8 +/- 3. In calf thymus DNA spiked with chemically synthesized 8-oxo-dGp to give an increment of 9 8-oxo-dG/10(6) dN, the added standard produced a significant increase with HPLC/ECD but not PPL. After spiking the DNA with 90 8-oxo-dG/10(6) dN, the added 8-oxo-dGp was detectable also with PPL, with a labeling efficiency of 65%. In order to investigate the role of ionizing radiation from 32P for the higher 8-oxo-dG levels in PPL, incubation times and amounts of radioactivity in the phosphorylation reaction with commercial dGp were increased, and external irradiation of commercial dG with 32P was investigated. All modifications resulted in higher values of 8-oxo-dG measured, but the effect was not large enough to fully explain the discrepancy between PPL and HPLC/ECD. Using [gamma-33P]ATP instead of [gamma-32P]ATP or adding [33P]phosphate to a 32P-PPL assay resulted in even higher levels of 8-oxo-dG measured. The increase in 8-oxo-dG levels during the PPL workup is attributed to the presence and oxidation of unmodified dGp in the reaction mixture. For a determination of true basal levels, the PPL method will have to be modified, including the removal of dGp prior to the phosphorylation reaction.
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PMID:Comparative analysis of 8-oxo-2' -deoxyguanosine in DNA by 32P- and 33P-postlabeling and electrochemical detection. 945 Apr 83

Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg(2+), not Ca(2+), and is inhibited by Zn(2+). The enzyme generates blunt ends or ends with 1-base 5'-overhangs possessing 5'-phosphate and 3'-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research.
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PMID:Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates. 1071 48


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