Micrococcal nuclease, chromatin structure

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]. 

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. 

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. 

Analysis of Chromatin Structure by Micrococcal Nuclease Digestion... 

This system has also been shown to be dependent on the secondary structure of DNA, the A, B, and Z forms reacting at different rates [150]. The likely explanation is that the faster reacting B DNA forms a more stable complex with the catalyst. This artificial DNase activity has also been compared with cleavage by micrococcal nuclease, and shown to recognize the same sites but not all those cleaved by DNase 1, again implying some local conformational preferences [151]. Chromatin structure has also been probed [152]. 

Brief digestion of chromatin with micrococcal nuclease results in the production of discrete units termed nucleosomes (nu bodies). On electron microscopy, these protein-DNA particles are approximately 10-13 nm long, linked by shorter (2-4 nm) DNA sequences. Further details of chromatin structure may be found in Chapter 7. 

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