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)

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.
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PMID:Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA. 78 11

Yeast mitochondrial DNA labelled in vitro by incubation of isolated mitochondria with DNA precursors exhibits skewed profiles on isopycnic CsCl gradients. The skew is not due to nuclear DNA nor to single-stranded mitochondrial DNA in the product labelled in vitro. Simultaneous labelling with [3H]dTTP and [14C]dGTP in vitro indicates a gradient of base composition in the DNA labelled in vitro. Thus, selective degradation of mitochondrial DNA occurs during incubation, converting large molecules having the mean density of mitochondrial DNA into smaller molecules of higher mean density and with higher G:T ratio. Similarly skewed distributions can also be produced by incubation of mitochondrial DNA labelled in vivo with the yeast mitochondrial fraction or with micrococcal endonuclease, an enzyme known to selectively hydrolyse (A plus T)-rich regions of DNA.
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PMID:Preferential digestion of (A plus T)-rich stretches of yeast mitochondrial DNA in isolated mitochondria. 110 Apr 8

We have examined the incorporation of biotinyl-11-deoxyuridine triphosphate (BiodUTP) into excision repair patches of UV-irradiated confluent human fibroblasts. Cells were reversibly permeabilized to BiodUTP with lysolecithin, and biotin was detected in DNA on nylon filters using a streptavidin/alkaline phosphatase colorimetric assay. Following a UV dose of 12 J/m2, maximum incorporation of BioUTP occurred at a lysolecithin concentration (80-100 micrograms/mL) similar to that for incorporation of dTTP. Incorporation of BiodUTP into repair patches increased with UV dose up to 4 and 8 J/m2 in two normal human fibroblast strains, while no incorporation of BiodUTP was observed in xeroderma pigmentosum (group A) human fibroblasts. The repair-incorporated biotin was not removed from the DNA over a 48-h period, and only slowly disappeared after longer times (approximately 30% in 72 h), while little of the biotin remained in cells induced to divide. Furthermore, the stability of the biotin in repaired DNA was unaffected by a second dose of UV radiation several hours after the biotin-labeling period to induce a "second round" of excision repair. Exonuclease III digestion and gap-filling with DNA polymerase I indicate that the majority of biotin-labeled repair patches (approximately 80%) are rapidly ligated in confluent human cells. However, the remaining patches were not ligated after a 24-h chase period, in contrast to dTTP-labeled repair patches. The BiodUMP repair label in both chromatin and DNA is preferentially digested by staphylococcal nuclease, preventing the use of this enzyme for nucleosome mapping in these regions.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of biotinylated repair regions in reversibly permeabilized human fibroblasts. 147 61

Isolated nuclei from pregnant rabbit mammary glands were labelled with [3H]dTTP under conditions for DNA synthesis and subsequently digested with micrococcal nuclease. Replicating chromatin was found to exhibit increased susceptibility towards the nuclease. Analysis of chromatin digestion products by sucrose density gradient centrifugation demonstrated the association of in vitro replicated DNA with nucleosomes. Furthermore, the distribution of DNA polymerizing activity was studied in isolated nuclease-digested mammary gland chromatin. About 90% of all recovered nuclear DNA polymerizing activity cosedimented with nucleosomal particles, mainly with mononucleosomes. The distribution of DNA polymerases alpha and beta in chromatin isolated from the mammary glands of pregnant and lactating rabbits was compared. In these physiological states the mononucleosome-associated DNA polymerase alpha activity varied in accordance with the rate of DNA synthesis.
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PMID:In vitro rabbit mammary gland chromatin replication studied by micrococcal nuclease digestion. 654 83

We have extended our permeable cell system for measuring DNA excision repair [Roberts, J. D., & Lieberman, M. W. (1979) Biochemistry 18, 4499-4505] so that steps of the repair process, beginning with incision and extending at least through the "rearrangement" of repaired nucleosomes which follows repair synthesis, all take place in permeable cells. In the revised protocol, human fibroblasts are made permeable, damaged with UV or chemicals in suspension, and incubated with a reaction mix containing ATP and the four deoxyribonucleoside triphosphates, one of which is labeled with 32P. By reducing the exogenous dNTP concentration to 3 microM and including 15 mM KCl in the reaction mixture, we have greatly reduced background incorporation in undamaged cells without significantly reducing repair synthesis. This permits us to measure repair synthesis without separating it from replicative synthesis by isopycnic centrifugation. Repair synthesis in this system is very similar to that occurring in intact cells: in response to DNA damage, nucleotides are incorporated into DNA of parental density (when analyzed by the BrdUrd density shift technique), incorporation increases with increasing DNA damage, synthesis is dependent on the presence of all four dNTPs, and the system accurately reflects the genetic UV repair deficiency of xeroderma pigmentosum (XP) cells. Furthermore, as has been observed in intact cells, repair-incorporated nucleotides in these permeable cells are initially overrepresented in staphylococcal nuclease sensitive regions of chromatin and are subsequently redistributed to give a nearly uniform distribution between nuclease-sensitive and -resistant regions. The UV dose curve of permeable cells differs somewhat from that of intact cells; however, the dose differs somewhat from that of intact cells; however, the dose curve for permeable cells treated with N-methyl-N-nitrosourea is very similar to that of intact cells. Repair synthesis in UV-damaged, permeable normal and XP cells is stimulated by addition of Micrococcus luteus UV endonuclease, indicating that the damaged DNA is accessible to exogenous repair enzymes and suggesting that incision, or an obligatory preincision step, is rate limiting for excision repair in these permeable cells. Repair synthesis in this system is inhibited by aphidicolin, but not by high levels of dideoxy-TTP, suggesting involvement of DNA polymerase alpha in excision repair. Novobiocin is also inhibitory alpha and the HeLa cell type II DNA topoisomerase.
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PMID:Characterization of deoxyribonucleic acid repair synthesis in permeable human fibroblasts. 709 2

Antibodies specific for acetylated histone H4 were used to examine the acetylation state of parental histones that segregate to newly replicated DNA. To generate newly replicated chromatin containing only segregated parental nucleosomes, isolated nuclei were labeled with [3H]TTP in vitro; alternatively, whole cells were labeled with [3H]thymidine in the presence of cycloheximide. Soluble chromatin was prepared by micrococcal nuclease digestion, and subjected to immunoprecipitation with "penta" antibodies (Lin et al., 1989). In sharp contrast to nucleosomes containing newly synthesized, diacetylated H4 (Perry et al., 1993), chromatin replicated in vitro was only marginally susceptible to immunoprecipitation. Control experiments established that bona fide acetylated chromatin was selectively immunoprecipitated by the same techniques and that segregated nucleosomes were not disassembled during treatment with "penta" antibodies. When replication was coupled to an in vitro histone acetylation system, the enrichment for segregated nucleosomes in the immunopellet increased approximately 3-fold, demonstrating that changes in the acetylation state of segregated histones can be detected immunologically and that parental histones on new DNA are accessible to acetyltransferases during, or immediately after, DNA replication. In vivo pulse-chase experiments, performed in the presence of cycloheximide, confirmed these results. Uptake experiments further established that concurrent histone acetylation did not alter the rate of DNA synthesis in vitro. Our results provide evidence that replication-competent chromatin is not obligatorily acetylated, and indicate that the acetylation status of segregated histones may be maintained during chromatin replication. The possible significance of this, with respect to the regulation of chromatin higher order structures during DNA replication, and the propagation of transcriptionally active vs inactive chromatin structures, is discussed.
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PMID:Parental nucleosomes segregated to newly replicated chromatin are underacetylated relative to those assembled de novo. 825 95