Chromatin structure mapping in Saccharomyces cerevisiae in vivo with DNase I

AUTOR(ES)

Wang, Xi

FONTE

Oxford University Press

RESUMO

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.

Documentos Relacionados

• High-resolution mapping of S1- and DNase I-hypersensitive sites in chromatin.
• Higher-order structure of Saccharomyces cerevisiae chromatin.
• High-resolution mapping of DNase I-hypersensitive sites of Drosophila heat shock genes in Drosophila melanogaster and Saccharomyces cerevisiae.
• DNase I-hypersensitive sites in the galactose gene cluster of Saccharomyces cerevisiae.
• Transcription-induced nucleosome 'splitting': an underlying structure for DNase I sensitive chromatin.