Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.21.1 (DNase)
 7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA polymerase from BHK-21/C13 cells were separated into two species, DNA polymerase I corresponding to the heterogeneous enzyme with sedimentation coefficient of 6-8S, and DNA polymerase II, corresponding to the enzyme with sedimentation coefficient of 3.3S. DNA polymerase I was purified 114-fold and DNA polymerase II 154-fold by a simple extraction procedure followed by column chromatography on phosphocellulose and gel filtration through Sephadex G-100. The purified enzymes differed markedly in respect of pH optimum, stimulation and inhibition by K+, Km for the deoxyribonucleoside 5'-triphosphates, stability to heating at 45 degrees C, and inhibition by N-ethylmaleimide. The preferred primer-template for both enzymes was "activated" DNA (DNA submitted to limited degradation by pancreatic deoxyribonuclease); native or thermally denatured DNA templates were relatively very poorly copied. When certain synthetic templates were tested, substantial differences were revealed between the two enzymes. Poly[d(A-T)] was poorly used by polymerase I but was superior to "activated" DNA for polymerase II. Poly[d(A)]-oligo[d(pT)10] was used efficiently by polymerase I but not by polymerase II. Poly(A)-oligo[d(pT)10] was not an effective primer-template although polymerase I could use it to a limited extent when Mn2+ replaced Mg2+ in the polymerase reaction and when the temperature of incubation was lowered from 37 degrees to 30 degrees C. When only one or two or three triphosphates were supplied in the reaction mixture, the activity of polymerase I was more severly diminished than that of polymerase II.
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PMID:Deoxyribonucleic acid polymerases of BHK-21/C13 cells. Partial purification and characterization of the enzymes. 23 80

The action of phenol on the products of partial digestion of calf thymus DNA by K2 DNAase causes a loss of 3--5% of the material passing into the phenol phase. This part of DNA can be regained in the aqueous phase by lowering both the temperature and the ionic strength. Among oligonucleotides up to 7 monomers in length, those which are soluble in phenol do not contain guanine residues. Phenol-soluble DNA fragments of the molecular weight of an order of 2000--50,000 appeared to be composed mainly of adenosine phosphate. They also contain some thymine and only traces of guanine and cytosine. Some longer A-T-rich fragments, even up to 5-10(6) daltons, were repeatedly found in the phenol phase, but their base composition has not been determined yet. The method presented here was found very convenient for the isolation of relatively large A-T-rich DNA fragments. The property of DNA or its fragments to dissolve in phenol seems to be dependent on an adequate primary structure, probably similar to that of poly (dA-dT).
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PMID:Isolation of A-T-rich fragments from calf thymus DNA using the phenol method. 84 48

A DNA-nuclear membrane complex has been isolated by two different methods from the nuclei of cultured mouse fibroblast (3T3) cells. One method, utilizing the detergent sarkosyl (sodium lauroyl sarkosinate), yields a DNA-nuclear membrane complex (the M band), which contains virtually all of the DNA in the nuclei. However, treatment of the M band by sonication, vortexing, or freeze-thaw reduces the amount of DNA in the complex by approximately 50-80%, depending upon the phase of the cell cycle from which the complex was extracted. The remaining DNA is tightly bound to the nuclear membrane and resists further shearing procedures. Over 90% of the choline-labeled phospholipid present in nuclei is also found in these sheared M bands. The percentage of DNA associated with the nuclear membrane varies during the cell cycle and correlates well with the onset, continuation, and cessation of DNA synthesis. Thus, although DNA-membrane complexes can be detected throughout the cell cycle, the percentage of DNA bound to membrane increases during late G1 and S and decreases during G2. In addition, there are distinct qualitative differences in the type of DNA present in the membrane fraction, with a more highly d(A-T) rich DNA being present in confluent (G0) cells than in cells during the S phase. This d(A-T) rich DNA may be related to the mouse satellite DNA identified by others. The M band can be separated into two DNA-nuclear membrane subfractions by centrifugation through a continuous sucrose gradient. The relative proportions of these two subfractions depend upon the percentage of sarkosyl present in the M band prior to centrifugation, with complete removal of sarkosyl resulting in a very large increase in the sedimentation velocity of the complex and in the formation of only one fraction. Evidence that this is a complex of DNA with membrane is given by the finding that DNA is dissociated from the complex with Pronase, deoxycholate, or high levels of sarkosyl. Removal of virtually all of the DNA with DNase from this rapidly sedimenting complex does not dissociate any of the phospholipid which still sediments rapidly as a single band. A second method, which yields a DNA-membrane fraction from nuclei, utilizes sedimentation of lysed nuclei to equilibrium in CsCl density gradients. This low-density CsCl fraction contains only 10-15% of the total DNA, but contains most of the nascent DNA, which may be chased into a membrane-free fraction. The DNA-membrane fraction from CsCl gradients possesses properties in common with the M-band fraction and can be converted into an M band. DNA membrane complexes from sucrose gradients, as well as the crude M-band preparation and a non-membrane-associated DNA fraction from nuclei can synthesize DNA in vitro without the addition of an external DNA template or DNA polymerase. In contrast to the activity in the non-membrane-associated DNA fraction, the membrane-associated polymerase activity is strongly stimulated by adenosine triphosphate and is unaffected by ethidium bromide...
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PMID:A nuclear membrane-associated DNA complex in cultured mammalian cells capable of synthesizing DNA in vitro. 99 Feb 45

The structure of eukaryotic chromatin has been investigated by isolating and analyzing the "accessible" DNA fraction of rat liver chromatin. This DNA fraction has been isolated by titrating the chromatin with the protese-resistant D isomer of polylysine to bind the "accessible" DNA sites. After removal of chromosomal proteins by digestion with pronase, all DNA not protected from attack by bound polylysine was removed by digestion with DNase. Even after exhaustive treatment with pronase and DNase approximately 30% of the chromatin DNA remains resistant to nuclease attack. Analysis of the isolated DNA shows it to be mainly double-stranded with an average size of 200-250 base pairs. The DNA is slightly A-T rich and contains both repetitive and "single-copy" nuleotide sequences. The results suggest that there are extensive regions in chromatin where the DNA is not tightly complexed with protein. Furthermore, the DNA of these regions is similar in gross properties to the DNA of the total genome.
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PMID:Isolation and characterization of the DNA fraction of rat liver chromatin which binds polylysine. 114 60

Porcine pancreatic DNase has been purified to homogeneity. The polypeptide exhibits a single band of Mr = 34,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a glycoprotein containing glucosamine. The results of end group analyses show leucine at the NH2 terminus and alanine at the COOH terminus. The enzymatic properties of the purified porcine DNase are very similar to those of bovine and ovine DNases. The sequence data on the tryptic and chymotryptic peptides derived from CNBr fragments of porcine DNase, along with the results of automated Edman degradation of the intact polypeptide and of the two largest CNBr fragments, indicate the complete amino acid sequence of porcine DNase to be as follows:L-R- I-A-F-N-I-R-T-F-G-E-T-K-M-S-N-A-T-S-N-Y-I-V-R-I-L-S-R-Y-D-I-A-L-I-Q- E-V-R-D-S-H-L-T-A-V-G-K-L-L-N-E-L-N-Q-D-D-P-N-N-Y-H-H-V-V-S-E-P-L-G-R- S-T-Y-K-E-R-Y-L-F-V-F-R-P-N-Q-V-S-V-L-D-S-Y-L-Y-D-D-G-C-E-P-C-G-N-D-T- F-N-R-E-P-S-V-V-K-F-S-S-P-F-T-Q-V-K-E-F-A-I-V-P-L-H-A-A-P-S-D-A-A-A-E- I-N-S-L-Y-D-V-Y-L-N-V-R-Q-K-W-D-L-Q-D-I-M-L-M-G-D-F-N-A-G-C-S-Y-V-T- T-S-H-W-S-S-I-R-L-R-E-S-P-P-F-Q-W-L-I-P-D-T-A-D-T-T-V-S-S-H-T-C-A-Y- D-R-I-V-V-A-G-P-L-L-Q-R-A-V-V-P-D-S-A-A-P-F-D-F-Q-A-A-F-G-L-S-Q-E-T- A-L-A-I-S-D-H-Y-P-V-E-V-T-L-K-R-A. The polypeptide consists of 262 amino acid residues. One of the two disulfide loops links Cys-101 and Cys-104 and the other Cys-173 and Cys-209. Two carbohydrate side chains are attached at Asn-18 and Asn-106.
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PMID:Purification, characterization, and the complete amino acid sequence of porcine pancreatic deoxyribonuclease. 378 4

Five DNA fragments have been cloned from yeast, chicken, and mouse DNA that titrate lac repressor in an Escherichia coli lac+ I+Z+ wild-type strain when on a multi-copy plasmid. The five repressor-binding sequences have been identified by DNA sequence determinations and DNase cleavage-inhibition patterns. They share the 14-base-pair symmetrical consensus sequence 5' T-G-T-G-A-G-C:G-C-T-C-A-C-A 3' (the colon represents the center of symmetry), which is an inverted repeat of 7 base pairs of the left half of the E. coli lac operator. A similar perfect palindromic DNA fragment--an 11-base-pair inverted repeat of the left half of the lac operator--was synthesized. The cloned synthetic DNA 5' G-A-A-T-T-G-T-G-A-G-C:G-C-T-C-A-C-A-A-T-T-C 3' binds lac repressor 8-fold more tightly than does wild-type E. coli lac operator DNA.
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PMID:Possible ideal lac operator: Escherichia coli lac operator-like sequences from eukaryotic genomes lack the central G X C pair. 636 30

A study on the sequence dependent DNA binding mode of DAPI has been carried out on pUC8 and the beta gal promoter region by restriction endonuclease and DNAase I protection experiments. A molecular model depicting drug interaction at the level of selected palyndromes has also been constructed that confirms the A-T sequence specificity of the compound. Experimental data indicate that the binding sites for RNA polymerase and cyclic AMP receptor protein (CRP) in the beta gal gene are privileged locales for DAPI interaction, a feature that explains impairment of transcription at this level. From a stereochemical view point, DAPI binding to DNA minor groove, while being incompatible with promoter unwinding in the open complex, may also disturb optimal contacts with proteins regulating RNA polymerase activity.
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PMID:A model for the sequence-dependent DNA binding of 4',6-diamidino-2-phenylindole (DAPI). 788 47

High mobility group protein HMG-I(Y) selectively binds to stretches of A.T-rich B-form DNA in vitro by recognition of substrate structure rather nucleotide sequence. Recognition of altered DNA structures has also been proposed to explain the preferential binding of this non-histone protein to four-way junction DNA as well as to restricted regions of DNA on random-sequence nucleosome core particles. Here we describe experiments that examine the influence of intrinsic DNA structure, and of structure imposed by folding of DNA around histone cores, on the binding of HMG-I(Y). As substrates for binding, we chose defined-sequence DNA molecules containing A.T-rich segments demonstrated previously to have very different structures in solution. These segments are either intrinsically bent (phase A.T tracts), flexible (oligo[d(A-T)]), or straight and rigid [oligo(dA).oligo(dT)]. DNase-I and hydroxyl radical footprinting techniques were employed to analyze protein binding to these DNAs either free in solution or when they were reconstituted into monomer or dinucleosomes in vitro. Results indicate that the DNA structure exerts a significant influence on HMG-I(Y) binding both when substrates are free in solution and when they are wrapped into nucleosomal structures. For example, when DNA is free in solution, HMG-I(Y) prefers to bind to the narrow minor groove of A.T sequences but sometimes also binds to certain GpC residues having narrowed major grooves that are embedded in such sequences. On the other hand, depending on the structure and/or orientation assumed by particular A.T-rich segments on the surface of reconstituted histone octamers, HMG-I(Y) binding site selection on individual nucleosomes differs considerably. Two observations are of particular importance: (i) HMG-I(Y) can preferentially bind to certain types of A.T-DNA located on the surface of nucleosomes; and (ii) HMG-I(Y) binding can induce localized alterations in the helical periodicity and/or rotational setting of DNA on the surface of some nucleosomes. The abilities of HMG-I(Y) suggests that in vivo the protein may play an important role in recognizing and altering the structure of localized regions of chromatin.
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PMID:Substrate structure influences binding of the non-histone protein HMG-I(Y) to free nucleosomal DNA. 866 99

The present report deals with the functional relationships among protein complexes which, when mutated, are responsible for four human syndromes displaying cancer proneness, and whose cells are deficient in DNA double-strand break (DSB) repair. In some of them, the cells are also unable to activate the proper checkpoint, while in the others an unduly override of the checkpoint-induced arrest occurs. As a consequence, all these patients display genome instability. In ataxia-telangiectasia, the mutated protein (ATM) is a kinase, which acts as a transducer of DNA damage signalling. The defective protein in the ataxia-telangiectasia-like disorder is a DNase (the Mre11 nuclease) that in vivo produces single-strand tails at both sides of DSBs. Mre11 is always present with the Rad50 ATPase in a protein machine: the nuclease complex. In mammals, this complex also contains nibrin, the protein mutated in the Nijmegen syndrome. Nibrin confers new abilities to the nuclease complex, and can also bind to BRCA1 (one of the two proteins mutated in familial breast cancer). BRCA1 has a central motif that binds with high affinity to cruciform DNA, a structure present in places where the DNA loops are anchored to the chromosomal axis or scaffold. The BRCA1 x cruciform DNA complex should be released to allow the nuclease complex to work in DNA recombinational repair of DSBs. BRCA1 also acts as a scaffold for the assembly of ATPases such as Rad51, responsible for the somatic homologous recombination. Loss of the BRCA1 gene prevents cell survival after exposure to cross-linkers. The BRCA1-RING domain is an E3-ubiquitin ligase. It can mono-ubiquitinate the FANCD2 protein, mutated in one of the Fanconi anemia complementation groups, to regulate it. Finally, during DNA replication, the nuclease complex and its activating ATM kinase are integrated in the BRCA1-associated surveillance complex (BASC) that contains, among others, enzymes required for mismatch excision repair. In short, the proteins missing in these syndromes have in common their BRCA1-mediated assembly into multimeric machines responsible for the surveillance of DNA replication, DSB recombinational repair, and the removal of DNA cross-links.
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PMID:Human syndromes with genomic instability and multiprotein machines that repair DNA double-strand breaks. 1250 2