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J Mol Biol. 2012 Mar 30;417(3):152-64. doi: 10.1016/j.jmb.2012.01.043. Epub 2012 Jan 30.

Micrococcal nuclease does not substantially bias nucleosome mapping.

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1
Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK. J.Allan@ed.ac.uk

Abstract

We have mapped sequence-directed nucleosome positioning on genomic DNA molecules using high-throughput sequencing. Chromatins, prepared by reconstitution with either chicken or frog histones, were separately digested to mononucleosomes using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both enzymes preferentially cleave internucleosomal (linker) DNA, although they do so by markedly different mechanisms. MNase has hitherto been very widely used to map nucleosomes, although concerns have been raised over its potential to introduce bias. Having identified the locations and quantified the strength of both the chicken or frog histone octamer binding sites on each DNA, the results obtained with the two enzymes were compared using a variety of criteria. Both enzymes displayed sequence specificity in their preferred cleavage sites, although the nature of this selectivity was distinct for the two enzymes. In addition, nucleosomes produced by CAD nuclease are 8-10 bp longer than those produced with MNase, with the CAD cleavage sites tending to be 4-5 bp further out from the nucleosomal dyad than the corresponding MNase cleavage sites. Despite these notable differences in cleavage behaviour, the two nucleases identified essentially equivalent patterns of nucleosome positioning sites on each of the DNAs tested, an observation that was independent of the histone type. These results indicate that biases in nucleosome positioning data collected using MNase are, under our conditions, not significant.

PMID:
22310051
PMCID:
PMC3314939
DOI:
10.1016/j.jmb.2012.01.043
[Indexed for MEDLINE]
Free PMC Article
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