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.

Keywords

DNA structure; cruciform DNA; genome organization

Published in

Molecular Plant
2016, volume: 9, number: 9, pages: 1328-1336

SLU Authors

• Gentry, Matthew

  • Department of Plant Biology, Swedish University of Agricultural Sciences
    

• Hennig, Lars

  • Department of Plant Biology, Swedish University of Agricultural Sciences
    

UKÄ Subject classification

Biochemistry and Molecular Biology
Developmental Biology

Publication identifier

• DOI: https://doi.org/10.1016/j.molp.2016.06.003

Permanent link to this page (URI)

https://res.slu.se/id/publ/81004