Hydrolysis Of Double-stranded DNA Research Articles

Reduced DNASE1 Gene Expression in Thyroid Autoimmunity

The enzyme DNASE1 plays an important role in the hydrolysis of double-stranded DNA and might be related to autoimmunity. Therefore, the authors hypothesized that patients with autoimmune thyroid disease show a reduced expression of the DNASE1 gene. DNASE1 mRNA was quantitatively analyzed in 20 patients (10 with Hashimoto's thyroiditis and 10 with Graves' disease) and 20 age- and sex-matched healthy controls by real-time reverse transcription PCR in a lightcycler using SYBR-Green-format. For relative quantification, the mRNA ratio of the DNASE1 gene to the house keeping gene β2-MICROGLOBULIN was used. The house keeping gene was proved not to be regulated by autoimmune thyroid disease. The interassay coefficient of variation for patients and controls was 22.2% and 15.6%, respectively, suggesting good reproducibility of measurements. The mean expression of the DNASE1 mRNA in patients was 0.52±0.22 (range 0.18-0.99) and in controls 0.95±0.22 (0.66-1.43). The expression level of the DNASE1 gene was strongly decreased in patients, amounting only 54.7% of that in controls (p<0.001). The lowered expression level in patients was not related to age or sex. This study demonstrated for the first time a downregulation of the DNASE1 mRNA expression in patients with autoimmune thyroid disease. This might result in degrading less DNA from dying cells, thereby promoting the development of thyroid autoimmunity.

DNase I – DNA interaction alters DNA and protein conformations

Human DNase I is an endonuclease that catalyzes the hydrolysis of double-stranded DNA predominantly by a single-stranded nicking mechanism under physiological conditions in the presence of divalent Mg and Ca cations. It binds to the minor groove and the backbone phosphate group and has no contact with the major groove of the right-handed DNA duplex. The aim of this study was to examine the effects of DNase I - DNA complexation on DNA and protein conformations. We monitored the interaction of DNA with DNase I under physiological conditions in the absence of Mg2+, with a constant DNA concentration (12.5 mmol/L; phosphate) and various protein concentrations (10-250 micromol/L). We used Fourier transfrom infrared, UV-visible, and circular dichroism spectroscopic methods to determine the protein binding mode, binding constant, and effects of polynucleotide-enzyme interactions on both DNA and protein conformations. Structural analyses showed major DNase-PO2 binding and minor groove interaction, with an overall binding constant, K, of 5.7 x 10(5) +/- 0.78 x 10(5) (mol/L)-1. We found that the DNase I - DNA interaction altered protein secondary structure, with a major reduction in alpha helix and an increase in beta sheet and random structures, and that a partial B-to-A DNA conformational change occurred. No DNA digestion was observed upon protein-DNA complexation.

Rapid site-selective hydrolysis of double-stranded DNA by use of Ce(IV)/EDTA and PNA bearing phosphate group

In order to hydrolyze double-stranded DNA efficiently at the target site, two pseudo-complementary peptide nucleic acids (pcPNAs) bearing phosphate group were combined with Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). The phosphate groups as metal-binding ligands were placed near the target site, and concentrated the Ce(IV) complex thereto. As the result, the site-selective hydrolysis was notably promoted, compared with the scission by the cutters involving unmodified pcPNAs.

Combination of S1 nuclease and PNA for site-selective hydrolysis of double-stranded DNA. -Comparison with the site-selective hydrolysis using Ce(IV)/EDTAY-

The potential of the combination of SI nuclease and pseudo-complementary PNA (pcPNA) for site-selective scission of double-stranded DNA has been investigated. Through strand invasion of two pcPNAs, single-stranded portions were formed in both strands of substrate DNA. In the initial stage of the enzymatic digestion, two scission fragments were obtained due to the hydrolysis at these two gap-like sites. On prolonged reactions, however, these products (as well as the substrate DNA) were further digested to smaller fragments. Under the conditions employed here, only Ce(IV)/EDTA is available for the preparation of desired fragments from double-stranded DNA.

DNA homology studies in Streptomyces using S1 nuclease.

The optimal reaction conditions for the determination of DNA-homology in Streptomyces species were established in the presence of formamide using S1 nuclease. The melting temperature of Streptomyces DNA was 90 degrees C in 0.42 M NaCl containing 20% formamide in which the denaturation was completed by boiling for 5 minutes. In the S1 reaction mixture consisting of 5 U of S1 nuclease, 0.168 M NaCl, 1 mM ZnSO4 and 8% formamide at pH 4.8, single-stranded DNA was hydrolyzed by more than 98%, while the hydrolysis of double-stranded DNA was less than 3%. From the analysis of homoduplex formation, the C0t1/2 was found at 20 hours, when a mixture of unlabeled DNA and index DNA was used at a ratio of 500:1.

A deoxyribonucleic acid binding protein from KB cells which inhibits deoxyribonuclease activity on single-stranded DNA.

A deoxyribonuclease inhibitor has been purified from KB cells by chromatography on single-stranded DNA-cellulose. Polyacrylamide gel electrophoresis showed the purified preparation to contain two major polypeptides in sodium dodecyl sulfate, with molecular weights of 72,000 and 65,000, but only one major band (with a molecular weight of approximately 140,000) after electrophoresis under nondenaturing conditions. The protein inhibits the hydrolysis of single-stranded DNA by KB DNase, DNase I, DNase II, and nuclease S1, but has no effect on the hydrolysis of double-stranded DNA by these enzymes. The inhibitor causes a reduction in the rate of hydrolysis of DNA by the deoxyribonuclease, probably by reducing the effective concentration of substrate.

Degradation of duplex DNA by S 1 nuclease from Aspergillus

Summary A nuclease from Aspergillus which selectively degrades single-stranded polynucleotides, S 1 , has been purified by DEAE-cellulose and Sephadex G-100 chromatography. The purified enzyme is still capable of degrading both OX174 RF and native calf thymus DNA. The hydrolysis of double-stranded DNA appears to be endonucleolytic as is single-strand cleavage.

An Endonuclease Induced after Infection of Escherichia coli with Bacteriophage T7: I. PURIFICATION AND PROPERTIES OF THE ENZYME

Abstract A deoxyribonuclease has been purified 1,800-fold from Escherichia coli infected with bacteriophage T7. The enzyme has an approximate molecular weight of 18,000, requires a divalent cation for activity, and is inhibited by high concentrations of RNA. The purified enzyme catalyzes an endonucleolytic cleavage of both single- and double-stranded DNA. The rate of hydrolysis of single-stranded DNA is 150-fold that of double-stranded DNA. The hydrolysis of single-stranded DNA results in the formation of acid-soluble oligonucleotides. No acid-soluble products have been observed during the hydrolysis of double-stranded DNA. Identity of the activities toward single- and double-stranded DNA is supported by identical sedimentation rates in sucrose gradients, reaction requirements, rates of heat inactivation, and genetic analysis.