title: A Structural Bisulfite Assay to Identify DNA Cruciforms.
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. This 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.
authors.affiliation: Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, 75007 Uppsala, Sweden.
authors.forename: Matthew
authors.initials: M
authors.lastname: Gentry
authors.affiliation: 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.
authors.forename: Lars
authors.initials: L
authors.lastname: Hennig
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D045566:DNA, Cruciform / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D018745:Genome, Plant / Q000235:genetics; D009707:Nucleosomes / Q000378:metabolism
chemical_list: D045566:DNA, Cruciform; D018744:DNA, Plant; D009707:Nucleosomes
keywords: DNA structure; cruciform DNA; genome organization