A Structural Bisulfite Assay to Identify DNA Cruciforms

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

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.

Keywords: DNA structure; cruciform DNA; genome organization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • DNA, Cruciform / genetics*
  • DNA, Plant / genetics*
  • Genome, Plant / genetics
  • Nucleosomes / metabolism

Substances

  • DNA, Cruciform
  • DNA, Plant
  • Nucleosomes