Nucleosome nuclease digestion

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. 

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. 

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. 

Histone chaperones bind histones and facilitate their proper deposition onto DNA by preventing nonspecific histone-DNA interactions (17). Two major histone chaperones are CAF-1 and NAP-1. CAF-1 localizes to the replication fork by binding PCNA and facilitates the deposition of histones H3 and H4 onto the newly synthesized DNA strands (18,19). Subsequently, NAP-1 facilitates the deposition of histones H2A and H2B to complete the nucleosome (20). Using in vitro nucleosome assembly and nuclease digestion mapping assays, it was shown that the periodic spacing of nucleosomes requires the function of ATP-dependent chromatin remodeling factors, such as the ACF/ISWI complex (16, 21). 

Structure of Nucleosomes The DNA component of nucleosomes Is much less susceptible to nuclease digestion than Is the linker DNA between them. If nuclease treatment Is carefully controlled, all the linker DNA can be digested, releasing individual nucleosomes with their DNA component. A nucleosome consists of a protein core with DNA wound around its surface like thread around a spool. The core is an octamer containing two copies each of histones H2A, H2B, H3, and H4. X-ray crystallography has shown that the octameric histone core is a roughly disk-shaped molecule made of interlocking histone subunits... 

Partial digestion by DNAase-I is widely used in the study of chromatin structure. Micrococcal nuclease (also called Staphyloccocal nuclease) makes double-stranded cuts initially in the linker DNA between nucleosomes, useful for mapping the positions of nucleosomes. Micrococcal nuclease digests must be interpreted carefully because this nuclease shows some sequence specificity when partial digestion of purified DNA is carried out as a control. In addition to these enzyme probes, there are chemical reagents that cut DNA. These reagents are much less sequence-dependent than even DNAase-I. Accessible DNA can also be labeled by methylating enzymes. 

In general, the remodeling of sperm chromatin that accompanies stage I decondensation involves the replacement of sperm-specific basic proteins with histones from the egg and results in the formation of nucleosomes. It now seems clear, at least in amphibian egg and Drosophila embryo extracts, that this remodeling is mediated by specific factors that participate in both assembly and disassembly processes. These changes in chromatin composition and structure can be analyzed by one- or two-dimensional polyacrylamide gel electrophoresis and micrococcal nuclease digestion. 

Preliminary results indicate that the DNA purified after micrococcal nuclease digestion of the enzyme—DNA complex activates more efficiently the DNA-free poly(ADP-ribose) polymerase than the total purified sDNA and much more than the purified nucleosomal core particles DNA (data not shown). Ohgushi et al. [8] and Benjamin and Gill [7] correlate the activation of poly(ADP-ribose) polymerase only to its binding to nicks or ends and not to a specific DNA sequence. Although our observations of poly(ADP-ribose) polymerase-sDNA complexes by electron microscopy and by polyacrylamide gel analysis do not exclude the possibUity that the enzyme is activated preferentially by internal nicks on sDNA fragments, they raise the question whether... 

Figure 36-2. Model for the structure of the nucleosome, in which DNA is wrapped around the surface of a flat protein cylinder consisting of two each of histones H2A, H2B, H3, and H4 that form the histone octamer. The 146 base pairs of DNA, consisting of 1.75 superhelical turns, are in contact with the histone octamer. This protects the DNA from digestion by a nuclease. The position of histone HI, when it is present, is indicated by the dashed outline at the bottom of the figure.

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... 

As to the stoichiometry of the H3-H4-DNA particle, two complexes were identified an H3-H4 tetramer and an H3-H4 octamer, each associated with about 140 base pairs of DNA. The complexing of 140 base pairs of DNA with H3 and H4 resulted in the formation of nucleosome-like particles, as observed by the EM, and reported to have an s20base pairs (Bina-Stein and Simpson, 1977 Bina-Stein, 1978). These results differ from those of Simon et al. (1978) who report that at least two complexes of H3 H4-DNA can be obtained upon reconstitution of H3, H4, and 150 bp DNA. In this experiment both an octamer and a tetramer of H3-H4 were found bound to 150 base pairs of DNA, having sM,w equal to 10.4 and 7.5 for the octamer and tetramer, respectively. The stoichiometry of the complexes obtained is dependent on the histone-to-DNA ratio. At low ratios of histone to DNA the predominant species contains an H3-H4 tetramer per 150 base pairs of DNA. At a histone-to-DNA ratio of 1 1 the octamer prevails. The nuclease and protease digestion experiments (Camerini-Otero et al., 1976 Sollner-Webb et al., 1976) were performed at a histone-to-DNA ratio of 0.5, conditions which for 140-base-pair DNA would lead primarily to a tetrameric complex. Therefore, it seems that a tetramer of H3 H4 is sufficient for the generation of nuclease-resistant fragments similar to those of complete nucleosomes. Upon addition of H2A and H2B to the tetrameric complex, nucleosomes are formed. Addition of H3-H4 to the tetrameric complex resulted in an octameric complex which is similar in compaction to nucleosomes. H3-H4 tetramers and octamers were similarly found complexed with about 140 base pairs of DNA upon reconstitution of H3-H4 with SV40 DNA. Both complexes were reported to be able to fold 140 base pairs of DNA (Thomas and Oudet, 1979). 

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]. 

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... 

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