DNA conformational transitions inferred from re-evaluation ofm|F_o| −D|F_c| electron-density maps

Abstract

Edited by G. J. Kleywegt, EMBL-EBI, Hinxton, EnglandKeywords: crystallographic heterogeneity; DNA; flexibility of phosphate backbone; Z-DNA; ZI/ZII transition. Conformational flexibility of DNA plays important roles in biological processes such as transcriptional regulation and DNA packaging etc. To understand the mechanisms of these processes, it is important to analyse when, where and how DNA shows conformational variations. Recent analyses have indicated that conventional refinement methods do not always provide accurate models of crystallographic heterogeneities and that some information on polymorphism has been overlooked in previous crystallographic studies. In the present study, the m|F o | À D|F c | electron-density maps of double-helical DNA crystal structures were calculated at a resolution equal to or better than 1.5 Å and potential conformational transitions were found in 27% of DNA phosphates. Detailed analyses of the m|F o | À D|F c | peaks indicated that some of these unassigned densities correspond to ZI $ ZII or A/B ! BI conformational transitions. A relationship was also found between ZI/ZII transitions and metal coordination in Z-DNA from the detected peaks. The present study highlights that frequent transitions of phosphate backbones occur even in crystals and that some of these transitions are affected by the local molecular environment.

A DNA structural alphabet provides new insight into DNA flexibility

“…The issue of incorrectly refined structures has been approached by Sunami et al (2017), who found that about a quarter of phosphate groups in higher resolution structures are actually disordered and may be positioned better in the electron density. The authors of the PDB_REDO web service (Joosten et al, 2009) offer an automated procedure to optimize crystallographic structure models deposited in the PDB.…”

Structural basis for designing an array of engrailed homeodomains

“…These results are indicative of the similarity between the DNA-binding modes of EHD[wt] and EHD [R53A]. We previously speculated that the R53A mutation would reduce the binding affinity without affecting the sequence specificity because it is unlikely to be involved in base-specific interactions (Sunami & Kono, 2019). Both the crystal structure and the functional analysis support this speculation.…”

“…To express (EHD[R53A]) 2 , we constructed the plasmid as described previously (Sunami & Kono, 2019). Briefly, we prepared an expression plasmid containing a DNA fragment encoding a polyhistidine tag-fused DNA-binding domain with the mutation and residues 453-512 of engrailed from Drosophila melanogaster.…”

“…DNA was subsequently extracted using a miniprep spin column (NIPPON Genetics) after culturing the cells in Luria-Bertani medium (BD) overnight at 37 C. The diversities of the libraries for wild-type EHD and for (EHD[R53A]) 2 were calculated to be 3.3 Â 10 6 and 3.0 Â 10 6 , respectively, which thoroughly covered the sequence space to be examined. The three pH3U3 plasmids containing the target sequences used in this work were previously isolated (Sunami & Kono, 2019). We introduced the pB1H2w5 plasmid library and the pH3U3 plasmid at a ratio of 1:20 into E. coli USO hisB À pyrF À rpoZ À cells by electroporation.…”

“…However, the 6 bp recognition length of EHD is not sufficient to precisely target a particular site within the large sequence space of a genome. We recently reported the construction of a molecular array consisting of two EHDs (EHD 2 ) and showed that EHD acts as a module, like the zinc finger and TAL effector (Sunami & Kono, 2019). A bacterial one-hybrid assay (B1H assay) showed that EHD 2 connected by a glycine linker recognizes only one 6 bp EHD target sequence in bacterial cells, which went against our expectation that it would bind to sequences with two EHD targets (12 bp).…”