Chromatin structure mapping in Saccharomyces cerevisiae in vivo with DNase I

Nucleic Acids Research
X Wang, R T Simpson

Abstract

Most methods for assessment of chromatin structure involve chemical or nuclease damage to DNA followed by analysis of distribution and susceptibility of cutting sites. The agents used generally do not permeate cells, making nuclear isolation mandatory. In vivo mapping strategies might allow detection of labile constituents and/or structures that are lost when chromatin is swollen in isolated nuclei at low ionic strengths. DNase I has been the most widely used enzyme to detect chromatin sites where DNA is active in transcription, replication or recombination. We have introduced the bovine DNase I gene into yeast under control of a galactose-responsive promoter. Expression of the nuclease leads to DNA degradation and cell death. Shorter exposure to the active enzyme allows mapping of chromatin structure in whole cells without isolation of nuclei. The validity and efficacy of the strategy are demonstrated by footprinting a labile repressor bound to its operator. Investigation of the inter-nucleosome linker regions in several types of repressed domains has revealed different degrees of protection in cells, relative to isolated nuclei.

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Citations

Jun 13, 2009·Chromosome Research : an International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology·Samantha SantangeloDontcho Z Staynov
Jun 8, 2002·Journal of Molecular Biology·Bernhard Suter, Fritz Thoma
Jul 13, 2007·Nucleic Acids Research·Fei XiaoWilliam T Garrard
Jun 17, 2003·Proceedings of the National Academy of Sciences of the United States of America·Christopher D CarvinMichael P Kladde
Oct 16, 2010·Cell Cycle·Rakesh Kumar SinghAkash Gunjan
Dec 18, 2013·ACS Nano·Michal Levy-SakinYuval Ebenstein
Sep 19, 2001·Yeast

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