A Structural Bisulfite Assay to Identify DNA Cruciforms Matthew Gentry, Lars Hennig  Molecular Plant  Volume 9, Issue 9, Pages 1328-1336 (September 2016) DOI: 10.1016/j.molp.2016.06.003 Copyright © 2016 The Author Terms and Conditions

Figure 1 Predicted Stem-Loop Structures of the RPS Element. For simplicity, only the top strand is shown. Weaker putative bonds in the loop structures are indicated by dashed lines. The NcoI site used for conversion-sensitive digests and key cytosine positions relating to bisulfite conversion graphs are indicated. Molecular Plant 2016 9, 1328-1336DOI: (10.1016/j.molp.2016.06.003) Copyright © 2016 The Author Terms and Conditions

Figure 2 Conversion-Sensitive Digests Showing Bisulfite Conversion at Predicted Single-Stranded Regions. PCR amplicons from converted chromatin covering FT palindrome 3 (upper) and the first RPS stem loop (lower) were digested with SnaBI and NcoI, respectively. In both cases, digestion is limited after bisulfite conversion, indicating that conversion has taken place. Molecular Plant 2016 9, 1328-1336DOI: (10.1016/j.molp.2016.06.003) Copyright © 2016 The Author Terms and Conditions

Figure 3 Increased Bisulfite Conversion Rates in Regions Covering Predicted Non-helical Secondary Structures. Values are averaged over two independent bisulfite conversions. At least 10 clones were sequenced per region and experiment. Primers for FT palindrome 11 were specific to the bottom strand so sequence numbers here are reversed. RPS, FT palindrome 3, and FT palindrome 11 all show significant cytosine conversion at regions corresponding to palindromic regions. Molecular Plant 2016 9, 1328-1336DOI: (10.1016/j.molp.2016.06.003) Copyright © 2016 The Author Terms and Conditions

Figure 4 Predicted Stem-Loop Structure in FT Corresponds to Sites of Increased Bisulfite Conversion. (A and B) Putative non-helical secondary structures are given for FT palindrome 3 (A) and FT palindrome 11 (B), showing the top strand only. Boxed regions indicate the approximate percentage of converted cytosines (grayed boxes) averaged over two independent conversions. At least 10 clones were sequenced per region and experiment. Molecular Plant 2016 9, 1328-1336DOI: (10.1016/j.molp.2016.06.003) Copyright © 2016 The Author Terms and Conditions

Figure 5 Identified Cruciform Structures Show Increased Susceptibility to S1 Nuclease Digestion. S1 sensitivity of regions after S1 nuclease serial digest. Regions covering FT palindrome 3 (FTp3), FTp11, and RPS show increased decay over control regions lacking palindromic regions (IGR, MAF5c and FTc), as well as MAF5p3, containing a palindromic element but with no conversion detected by the structural bisulfite assay. Shown are total amounts of DNA-digested decay constants and their SE derived from a regression of Y = y0 +ae−λxt, where Y is the relative amount of remaining test region DNA, y0 is the S1-resistant fraction of DNA, a is the S1-sensitive fraction, t is the reaction time, and x is the S1 concentration, to the experimental data. Molecular Plant 2016 9, 1328-1336DOI: (10.1016/j.molp.2016.06.003) Copyright © 2016 The Author Terms and Conditions

Figure 6 The FT Palindrome 3 Structural Sequences Are Conserved Across Two Arabidopsis Accessions and Eight Other Brassicaceae. Hyperconserved regions S1 and S2 identified by Adrian et al. (2010) are indicated at the bottom. The mispaired G residue in the Col FT palindrome 3 structure is indicated by an asterisk. The loop region is indicated by the central box and proposed stem regions by lines at the top of the alignment. Dashed lines indicate the variable regions around the mispaired G, or on the corresponding side of the stem structure. Molecular Plant 2016 9, 1328-1336DOI: (10.1016/j.molp.2016.06.003) Copyright © 2016 The Author Terms and Conditions