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Mol Plant. 2016 Sep 6;9(9):1328-1336. doi: 10.1016/j.molp.2016.06.003. Epub 2016 Jun 29.

A Structural Bisulfite Assay to Identify DNA Cruciforms.

Author information

1
Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, 75007 Uppsala, Sweden.
2
Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, 75007 Uppsala, Sweden. Electronic address: lars.hennig@slu.se.

Abstract

In the half century since the discovery of the double-helix structure of DNA, it has become increasingly clear that DNA functionality is based on much more than its sequence in a double-helical structure. Further advances have highlighted the importance of additional aspects of DNA structure: its packaging in the higher order chromatin structure, positioning of nucleosomes along the DNA, and the occurrence of non-helical DNA structures. Of these, the latter has been problematic to prove empirically. Here, we describe a method that uses non-denaturing bisulfite sequencing on isolated Arabidopsis thaliana nuclei to determine the location of cytosines positioned outside the double helix as a result of non-B-form DNA structures. We couple this with computational methods and S1 nuclease digest to reliably identify stable, non-B-form, cruciform structures. ThisĀ enables us to identify a palindrome in the promoter of FLOWERING LOCUS T that forms a stable non-B-form structure. The stronger conservation of the ability to form a non-helical secondary structure than of the sequence suggests that this structure is biologically relevant.

KEYWORDS:

DNA structure; cruciform DNA; genome organization

PMID:
27375204
DOI:
10.1016/j.molp.2016.06.003
[Indexed for MEDLINE]
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