DNases micrococcal nuclease

Other substrates for spleen exonuclease are the p-nitrophenyl esters of nucleoside-3 -phosphates and bis(p-nitrophenyl) phosphate, which is split only very slowly. These substrates are also split by enzymes having quite different natural substrates (Table I) (80-87). In fact, not only phosphodiesterases, in a broad sense, such as acid DNase, micrococcal nuclease, spleen and venom exonucleases, and cyclic phosphodiesterase but also enzymes such as nucleoside phosphoacyl hydrolase and nucleoside polyphosphatase split these substrates. As pointed out by Spahr and Gesteland (86), this may be explained by the fact that these substrates are not true diesters but rather mixed phosphoanhydrides because of the acidic character of the phenolic OH. It is evident that the use of the synthetic substrates, advocated by Razzell (3) as specific substrates for exonucleases, may be very misleading. Table II shows the distinctive characters of three spleen enzymes active on bis(p-nitrophenyl) phosphate which are present in the crude extracts from which acid exonuclease is prepared. 

Fig. 8. Schematic for the micrococcal nuclease attack to (a) breathing ds-DNA, (b) dista-mycin-bound DNA, and (c) Zn2+-cyclen derivative-bound DNA. Arrows and dashed arrows, respectively, indicate successful and failed enzyme hydrolysis. Reproduced with permission from Ref. (37). Copyright 1999, American Chemical Society.

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. 

The nucleosome is composed of 200 base pairs of DNA and an octamer of the histones H2A, H2B, H3, and H4 as well as histone HI (Komberg, 1974, 1977). Nucleosomes can be obtained by mild digestion of chromatin with micrococcal nuclease (Noll, 1974a Axel, 1975), followed by fractionation on a sucrose gradient. Further digestion of the nucleosomes results in the formation of nucleosome core particles composed of 145 base pairs of DNA and an octamer of the histones H2A, H2B, H3, and H4 (Rill and Van Holde, 1973 Sollner-Webb and Felsenfeld, 1975 Axel, 1975 Bakayev et al., 1975 Whitlock and Simpson, 1976 Noll and Komberg, 1977). The DNA piece thus excised is called linker DNA which serves as a link... 

Similar results were obtained from reconstitution experiments with DNA and a non-cross-linked octamer (Thomas and Butler, 1978). Nucleosome-like particles were observed in the EM and a pattern of histone cross-linking comparable to that of native chromatin was obtained. However, only 140-base-pair repeats were obtained upon micrococcal nuclease digestion instead of 200-base-pair repeats obtained for native rat liver chromatin (Noll and Komberg, 1977). This indicates that, in the absence of HI, only core particles can be reconstituted. Nevertheless, these studies with both cross-linked and reassembled un-cross-linked histones demonstrate that the octamer is a complete biological functional unit retaining the information for folding the DNA around the histone core. 

Nucleosomes core particles containing H2A only have 118 base pairs of DNA incorporated compared to the canonical nucleosomes protecting about 147 base pairs from micrococcal nuclease (Bao et al. 2004). These nucleosomes are more flexible in structure and might facilitate passage of RNA polymerase II. However, the function of this histone variant in mammalian cells is not fully understood. As... 

Antibiotic Mg complex induced alteration in the ultrastructural changes in the native and HI depleted chromatin were monitored by thermal melting analysis, polyacrylamide gel mobility assay, dynamic light scattering experiments and transmission electron microscopic studies. Micrococcal nuclease digestion is the biochemical probe to assess the accessibility of the antibiotic Mg + complexes to nucleosomal DNA. 

The nucleosome is the fundamental repeating structural unit of chromatin. It is composed of two molecules of the core histones H2A, H2B, H3, H4, approximately two superhelical turns of double-stranded DNA, and linker histone HI (H5). In addition to biochemical studies, the existence of the nucleosome was established in electron micrographs (Fig. la) [1,2], and the name nucleosome, coined to incorporate the concept of the spherical nu-bodies [3]. Micrococcal nuclease limit digestion of chromatin established the nucleosome core particle (NCP) as the portion of the nucleosome containing only the core histones surrounded by 1.75 superhelical turns of double-stranded DNA [4,5]. 

Several lines of evidence indicate that CENP-A replaces conventional H3 in the nucleosome. Biochemical studies showed that CENP-A co-sediments with nucleo-some core particles [7] and a genetic analysis indicates an interaction between Cse4p, the CENP-A of Saccharomyces cerevisiae, and H4 [16,17]. A recent study with CENP-A purified from HeLa cells or expressed in bacteria showed that it can substitute for conventional H3 in nucleosome reconstitution [18]. Reconstituted CENP-A-containing nucleosomes appear to contain the other core histones in appropriate stoichiometry. However, they did not strongly protect 146 bp of core DNA from micrococcal nuclease, suggesting that CENP-A may significantly alter some aspects of the core nucleosome structure. 

It is obvious that the single-molecule approaches can reveal features of the dynamics of chromatin assembly and its force dependence that could not be observed using the standard biochemical assays to study assembly following changes in superhelicity in closed circular DNA molecules, looking at changes in protection against enzymes like micrococcal nuclease, or the accessibility of restriction enzymes. 

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... 

This hydrolase, officially known as micrococcal nuclease [EC 3.1.31.1 (formerly 3.1.4.7)], catalyzes endonucleolytic cleavage of single-Zdouble-stranded DNA (as well as RNA) to form 3 -phospho-mononucleotide and 3 -phospho-oligonucleotide end-products. The enzyme also displays exonucleolytic activity. 

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). 

Wechter (34) and Richards et al. (35) showed that venom exonuclease is capable of hydrolyzing dinucleoside monophosphates w ith one or both nucleosides containing arabinose. Even though only four different sugars have been tested, it would appear that venom exonuclease is totally blind to sugar, at least in the qualitative sense. Other enzymes capable of hydrolyzing DNA and RNA are incapable of hydrolyzing derivatives of arabinose, e.g., micrococcal nuclease (SO). 

Marx, K.A. and Reynolds, T.C. (1982) Spermidine-condensed phi XT74 DNA cleavage by micrococcal nuclease toms cleavage model and evidence for unidirectional circumferential DNA wrapping. Proc. Natl. Acad. Sci. USA, 79, 6484-6488. 

Micrococcal nuclease Staphylococcus aureus RNA and DNA endonuclease splits d bonds in areas rich in adenosine, uracil, and thymine... 

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