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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)

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

Topological knots can be formed in vitro by incubating covalently closed double stranded DNA and purified topoisomerase II from the yeast Saccharomyces cerevisiae in an ATP-dependent reaction. Knotting production requires a starting enzyme/DNA mass ratio of 1. Analysis of knotted DNA was carried out by using both one- and two-dimensional agarose gel electrophoresis. The knots generated are efficiently untied, and give relaxed DNA rings, by catalytic amounts of topoisomerase II, but not by topoisomerase I. Time course analysis shows the knotting formation over relaxed and supercoiled DNA. When supercoiled DNA was used as a susbtrate, knots appear immediately whereas no transient relaxed rings were observed. The cell-free extract from Xenopus oocytes S-150 cannot assemble nucleosomes on knotted DNA templates as revealed by topological and micrococcal nuclease analysis. Nevertheless, the presence of knotted DNA templates does not inhibit the assembly over the relaxed plasmid. Finally, a pretreatment of knotted DNA with trace amounts of topoisomerase II before the addition of the S-150 yields a canonical minichromosome assembled in vitro. Taking into account these results, I suggest a mechanism of chromatin assembly regulation directed by topoisomerase II.
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PMID:DNA knotting abolishes in vitro chromatin assembly. 866 64

We have analyzed the chromatin structure of mouse ribosomal RNA genes (rDNA) by partial digestion of genomic DNA with micrococcal nuclease (MNase), DNase I and identified hypersensitive sites by indirect end-labeling. This analysis has revealed defined regions of nuclease hypersensitivity in the intergenic spacer which in turn coincide with regulatory elements. Hypersensitive sites map to the transcription initiation site, the enhancer repeats, the spacer promoter and two sequence elements which coincide with amplification-promoting sequences. Analysis of the DNA curvature by computer modeling uncovered a striking correlation between sequence-directed structural features of regulatory regions and the position of nuclease hypersensitive sites. Moreover, we demonstrate that nucleosomes are specifically positioned upstream and downstream of the transcription start site. In vitro studies using chromatin assembled in the presence of Drosophila embryo extracts show that binding of the transcription termination factor TTF-I to the upstream terminator mediates this specific nucleosome positioning at the rDNA promoter in an ATP- dependent fashion.
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PMID:Structural analysis of mouse rDNA: coincidence between nuclease hypersensitive sites, DNA curvature and regulatory elements in the intergenic spacer. 901 89

Nuclease hypersensitive sites exist in vivo in the chromatin of the integrated human immunodeficiency virus (HIV)-1 proviral genome, in the 5'-long terminal repeat (LTR) within the promoter/enhancer region near Sp1 and NFkappaB binding sites. Previous studies from the Kadonaga and Jones laboratories have shown that Sp1 and NFkappaB can establish hypersensitive sites in a truncated form of this LTR when added before in vitro chromatin assembly with Drosophila extracts, thus facilitating subsequent transcriptional activation of a linked reporter gene upon the association of additional factors (Pazin, M. J., Sheridan, P. L., Cannon, K., Cao, Z., Keck, J. G., Kadanaga, J. T., and Jones, K. A. (1996) Genes & Dev. 10, 37-49). Here we assess the role of a full-length LTR and 1 kilobase pair of downstream flanking HIV sequences in chromatin remodeling when these transcription factors are added after chromatin assembly. Using Xenopus laevis oocyte extracts to assemble chromatin in vitro, we have confirmed that Sp1 and NFkappaB can indeed induce sites hypersensitive to DNase I, micrococcal nuclease, or restriction enzymes on either side of factor binding sites in chromatin but not naked DNA. We extend these earlier studies by demonstrating that the process is ATP-dependent when the factors are added after chromatin assembly and that histone H1, AP1, TBP, or Tat had no effect on hypersensitive site formation. Furthermore, we have found that nucleosomes upstream of NFkappaB sites are rotationally positioned prior to factor binding and that their translational frame is registered after binding NFkappaB. On the other hand, binding of Sp1 positions adjacent downstream nucleosome(s). We term this polar repositioning because each factor aligns nucleosomes only on one side of its binding sites. Mutational analysis and oligonucleotide competition each demonstrated that this remodeling required Sp1 and NFkappaB binding sites.
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PMID:In vitro chromatin assembly of the HIV-1 promoter. ATP-dependent polar repositioning of nucleosomes by Sp1 and NFkappaB. 921 15

32P-Postlabelling methods have been investigated for the analysis of the oxidative DNA damage lesion 8-oxoguanine. The extent of digestion of commercially available calf thymus DNA and an 8-oxo-2'-deoxyguanosine-3'-monophosphate (8oxodGp) containing oligonucleotide to 2'-deoxynucleotide-3'-monophosphates, using calf spleen phosphodiesterase and micrococcal nuclease, was determined by HPLC. The extent of unmodified nucleotide release from DNA, and the extent of 8oxodGp released from the oligomer did not increase between 1 and 16 h of incubation at 37 degrees C. Normal nucleotide release from DNA was found to be quantitative under these conditions, and 8oxodGp release from the oligomer was in the range of 84-91%. RNA contamination in DNA prepared for 32P-postlabelling severely compromised 8oxodGp analysis. Guanosine-3'-monophosphate (Gp) was found to exhibit similar chromatographic and electrophoretic properties to 8oxodGp and as such compromised both 8oxodGp isolation in enrichment steps and subsequent resolution of the 32P-labelled bisnucleotides by TLC. The effect of ribonuclease A, T1 and T2 was investigated and a combination of A + T1 was found to reduce Gp contamination in DNA samples to levels which no longer interfered with 8oxodGp analysis. We have successfully applied an HPLC enrichment protocol to the analysis of 8oxodGp in calf thymus DNA. Since determination of damage levels in human samples is often restricted by the amount of DNA available for analysis, a novel capillary electrophoresis (CE) technique for the enrichment of 8oxodGp has been developed. The advantage of CE is that it can achieve resolution of 8oxodGp and unmodified deoxynucleotides from much smaller samples and minimises the amount of [gamma-32P]ATP necessary for the analysis.
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PMID:32P-postlabelling approaches for the detection of 8-oxo-2'-deoxyguanosine-3'-monophosphate in DNA. 928 92

We have compared 70-kDa heat shock cognate protein (Hsc70) isolated from bovine brain with recombinant wild type protein and mutant E543K protein (previously studied as wild type in our laboratory). Wild type bovine and recombinant protein differ by posttranslational modification of lysine 561 but interact similarly with a short peptide (fluorescein-labeled FYQLALT) and with denatured staphylococcal nuclease-(Delta135-149). Mutation E543K results in 4. 5-fold faster release of peptide and lower stability of complexes with staphylococcal nuclease-(Delta135-149). ATP hydrolysis rates of the wild type proteins are enhanced 6-10-fold by the addition of peptide. The E543K mutant has a peptide-stimulated hydrolytic rate similar to that of wild type protein but a higher unstimulated rate, yielding a mere 2-fold enhancement. All three versions of Hsc70 possess similar ATP-dependent conformational shifts, and all show potassium ion dependence. These data support the following model: (i) in the presence of K+, Mg2+, and ATP, the peptide binding domain inhibits the ATPase; (ii) binding of peptide relieves this inhibition; and (iii) the E543K mutation significantly attenuates the inhibition by the peptide binding domain and destabilizes Hsc70-peptide complexes.
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PMID:Destabilization of peptide binding and interdomain communication by an E543K mutation in the bovine 70-kDa heat shock cognate protein, a molecular chaperone. 934 24

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

We have analyzed the effect of the chaperonin GroEL on the refolding kinetics of staphylococcal nuclease and its three mutants by stopped-flow fluorescence measurements. It was found that a transient folding intermediate of staphylococcal nuclease was tightly bound to GroEL and refolded in the GroEL-bound state without releasing the non-native protein in solution, and the refolding rate in the GroEL-bound state was 0.01 s-1. The GroEL-affected refolding of the nuclease appears to be in decided contrast to that of apo-alpha-lactalbumin reported in our previous study, wherein alpha-lactalbumin was shown to be more weakly bound by GroEL and to refold in the free state in solution. In spite of the apparent difference between the proteins, the GroEL-affected refolding reactions of both the proteins can be represented by a common unified reaction scheme. On the basis of this scheme, the binding constant between the nuclease intermediate and GroEL was estimated to be larger than 10(9) M-1. The stoichiometry of binding of the nuclease and its mutants to GroEL was found to be two (nuclease/GroEL 14-mer). The increase in ionic strength resulted in a weakening of the interaction between the nuclease and GroEL, which was attributed to a weakening of the electrostatic attraction between the two proteins as a result of electrostatic screening by ions. Although ATP was found to accelerate the GroEL-affected refolding of the nuclease, the refolding rate was still far from the rate of the free refolding. The free refolding behavior of the nuclease and its mutants was restored in the presence of the cochaperonin GroES and ATP.
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PMID:Refolding kinetics of staphylococcal nuclease and its mutants in the presence of the chaperonin GroEL. 953 91

The human SWI/SNF complex remodels nucleosome structure in an ATP-dependent manner, although the nature of this change has not been determined. Here we show that hSWI/SNF and ATP generate an altered nucleosomal structure that is stable in the absence of SWI/SNF. This product has an altered sensitivity to digestion by DNAse, restriction enzymes, and micrococcal nuclease, and an increased affinity for GAL4. It has the same protein composition but is approximately twice the size of a normal nucleosome. Incubation of the altered nucleosome with hSWI/SNF converts this structure back to a standard nucleosome in an ATP-dependent process. These results suggest that hSWI/ SNF acts by facilitating an exchange between normal and altered, more accessible, nucleosome conformations.
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PMID:Human SWI/SNF interconverts a nucleosome between its base state and a stable remodeled state. 967 23

Mcm (minichromosome maintenance) proteins are important components of the eukaryotic replication initiation apparatus. We investigate the binding of human Mcm proteins to HeLa cell chromatin using micrococcal nuclease as a tool. In previous work we prepared chromatin under low ionic strength conditions. The use of a low salt buffer was necessary to prevent the dissociation of Mcm proteins. Here we use chromatin prepared at more physiological salt concentrations (100 mM NaCl) following the procedure of Fujita et al. (J. Biol. Chem. 272, 10928-10935; 1997) who had shown that ATP stabilizes the interaction of Mcm proteins with chromatin. We show here that micrococcal nuclease released Mcm proteins early during the digestion process suggesting that Mcm proteins reside on chromatin sites which are more open to nuclease attack than bulk chromatin. Released Mcm proteins sedimented through glycerol gradients as a multiprotein complex comprising several of the six known human Mcm proteins.
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PMID:Mobilization of chromatin-bound Mcm proteins by micrococcal nuclease. 979 52


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