Nucleases DNase

One of the very early research tools that were used to study the nucleosomal state of active genes were the nucleases, DNase I and Micrococcal nuclease. With the development of protocols for the isolation of nuclei from cells, it was possible to add these reagents to probe the accessibility of DNA. DNase I makes single nicks in double stranded DNA and when the DNA is associated with histones within the nucleosome, the DNA is extensively protected. Those nicks that are observed are found to occur only after extensive digestion and are limited to the outside surface of the DNA in 10 base increments [7,8]. Weintraub and Groudine in 1976 [9] first used this nuclease and observed that when nuclei from chicken erythrocytes were treated with DNase I, the active /1-globin gene was preferentially... 

Three levels of protein-nucleic acid recognition have been observed. Nature provides three examples of protein-nucleic acid interactions which we shall consider. The nucleic acid component can be (1) a single nucleotide, e.g., a coenzyme or a substrate, (2) a single-stranded DNA or RNA as in ribonucleases A and T, or (3) a double-stranded DNA or RNA as in the highly specific complexes with repressors in the tRNA-synthetase complex, or in the unspecific nuclease DNase I. 

In vivo, cleavage of P-0 bonds are performed by enzymes such as phosphatases, phosphodiesterases, phosphohydrolases, nucleases, DNases and RNases (see Section 13.1.1). In vitro, cleavage of a P- O bond is often a trivial synthetic step. Even for an easy step, enzymes attract increasing attention. The enzymatic reactions are preferred when regio- or stereoselectivity is required, and when the substrates are temperature or pH sensitive. Many phosphate analogs have been tested as substrates of enzymes that hydrolyze phosphoryl groups. These analogs are often accepted as substrates for the enzymes, and such reactions could be synthetically valuable. Typical examples are presented in the tables. 

Metal ions also participate in the functioning of other nucleases, although the structural details of their participation are not nearly as established as those for staphylococcal nuclease. DNAse I also requires Ca for its catalytic activity.SI endonuclease, mung bean nuclease, and Physarum polycephalem nuclease require zinc ion either as cofactors or intrinsically for nuclease activity, and the restriction enzyme EcoRI may also require intrinsically bound zinc ion. In terms of how the zinc ion might function in these enzymes, one can look both to staphylococcal nuclease and to bacterial alkaline phosphatase for some... 

Yes, the incorporation of nucleoside triphosphates into an acid-insoluble form is indicative of the presence of a polymerase. The polymerase is likely a DNA polymerase because dNTPs, and not NTPs, were used to form product. Further evidence for a DNA polymerase was that the radiolabeled product was destroyed by a nuclease, DNase, specific for hydrolyzing DNA, and not by one specific for RNA hydrolysis. Additionally, NaOH, which destroys RNA but not DNA, did not destroy the radiolabeled product. Pretreatment of the extract with the two hydrolytic enzymes demonstrated that the enzyme depends on an RNA and not a DNA template for its activity. Thus, this enzyme is an RNA-dependent DNA polymerase. No such enzyme had been observed previously in a cell, and this demonstration, along with similar findings by Howard Temin, of its existence in an RNA tumor virus caused a revision of Francis Crick s central dogma of molecular biology, which stated that information flowed from DNA to RNA to proteins. The demonstration of this RNA-dependent DNA polymerase suggested that in some cases information could flow from RNA to DNA. (This question was derived from D. Baltimore. Viral RNA-dependent DNA polymerase. Nature 226 [1971]1209-1213.)... 

As a nonspecific nuclease, DNase I is extensively used in molecular biology and recombinant DNA technology. The following examples are given to illustrate the contexts and strategies for using DNase I. 

Fike most enzymes (see Chapter 14), nucleases exhibit selectivity or specificity for the nature of the substance on which they act. That is, some nucleases act only on DNA (DNases), while others are specific for RNA (the RNases). Still... 

The histone core protects the DNA bound to the nucleosome from digestion by pan-creatic deoxyribonuclease (DNase) I or micrococcal nuclease. Nucleases, however, will cleave the linker DNA that connects the nucleosome subunits to one another. 

Nuclease digestion of targets by endogenous RNase or DNase or by contaminating RNase. 

Wood, W.I. and Felsenfeld, G. (1982) Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II. J. Biol. Chem. 10 257(13), 7730-7736. 

Several hydrolases—particularly ribo-nuclease (RNAse) and deoxyribonuclease (DNAse)—break down the nucleic acids contained in food. 

To explain the enzymology of DNA replication, we first introduce the enzymes that degrade DNA rather than synthesize it. These enzymes are known as nucleases, or DNases if they are specific for DNA rather than RNA. Every cell contains several different nucleases, belonging to two broad classes exonucleases and endonucleases. Exonucleases degrade nucleic acids from one... 

A multitude of nucleases cleave DNA, single- or double-stranded. They range from the pancreatic digestive enzyme DNase I through specialized nucleases that function during DNA repair and the hundreds of restriction endonucleases that have become so valuable in modern laboratory work. Some nucleases leave a 3 -phosphate ester at a cut end in a DNA chain, while others leave a 5 -phosphate end.824 Many nucleases are dealt with in later chapters. Only a few will be mentioned here. 

Microbial RNases with known substrate specificity are listed in Table XII. Nucleases with DNase activity are not included. It should be noted here that the lists of RNases in both animal and plant kingdoms are presented in the monograph by Privat de Garilhe (4) and in a chapter by E. A. Barnard in Annual Review of Biochemistry (1969) (135). 

Among endonucleases which hydrolyze DNA one seldom finds an enzyme that attacks double-stranded and single-stranded substrates with equal ease. If the enzyme shows preference for double-stranded substrates (as DNase I does) autoretardation is observed. This decrease in the reaction rate is caused by the gradual disappearance of the preferred, double-stranded substrate and an increase in the concentration of less susceptible, single-stranded substrate. Differences in rates between the early and terminal phases of the reaction of the order of 1000-fold have been described (< ). The opposite case, autoacceleration, is seen with those enzymes that show preference for the single-stranded structure, e.g., micrococcal nuclease (7). 

Many DNases are known to be activated by a divalent cation. However, only from the work of Bollum (11) did it become clear that the nature of the cation may qualitatively change the specificity of the enzyme toward adjacent bases. Quantitative changes in the requirements for the divalent cation (10) have been observed during different stages of the same reaction, e.g., micrococcal nuclease (7) where the increased Ca2+ concentration causes a decrease in the average size of the terminal product. 

This discovery of Bollum (11) makes obsolete a number of previous excellent studies including those on mutual interdependence between concentrations of divalent metal, monovalent metal, hydrogen ion, and substrate. Unless the bond affected by the metal in question is specified, an overall rate represents a number with little value. The problem is further complicated by the suspected (by analogy to other nucleases) quantitative changes in requirements for metal ions at different stages of the reaction. So far no such data are available for DNase I. One is tempted to add, luckily, because in view of the uncertainty of qualitative effects such data would hardly be expected to have a long survival time. 

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