Halogen bonds (X-bonds) are shown to be geometrically perpendicular to and energetically independent of hydrogen bonds (H-bonds) that share a common carbonyl oxygen acceptor. This orthogonal relationship is accommodated by the in-plane and out-of-plane electronegative potentials of the oxygen, which are differentially populated by H- and X-bonds. Furthermore, the local conformation of a peptide helps to define the geometry of the H-bond and thus the oxygen surface that is accessible for X-bonding. These electrostatic and steric forces conspire to impose a strong preference for the orthogonal geometry of X- and H-bonds. Thus, the optimum geometry of an X-bond can be predicted from the pattern of H-bonds in a folded protein, enabling X-bonds to be introduced to improve ligand affinities without disrupting these structurally important interactions. This concept of orthogonal molecular interactions can be exploited for the rational design of halogenated ligands as inhibitors and drugs, and in biomolecular engineering.

Mef2 genes encode highly conserved transcription factors involved in somitic and cardiac mesoderm development in diverse bilaterians. Vertebrates have multiple mef2 genes. In mice, mef2c is required for heart and vascular development. We show that a zebrafish mef2c gene (mef2ca) is required in cranial neural crest (CNC) for proper head skeletal patterning. mef2ca mutants have head skeletal phenotypes resembling those seen upon partial loss-of-function of endothelin1 (edn1). Furthermore, mef2ca interacts genetically with edn1, arguing that mef2ca functions within the edn1 pathway. mef2ca is expressed in CNC and this expression does not require edn1 signaling. Mosaic analyses reveal that mef2ca is required in CNC for pharyngeal skeletal morphogenesis. Proper expression of many edn1-dependent target genes including hand2, bapx1, and gsc, depends upon mef2ca function. mef2ca plays a critical role in establishing the proper nested expression patterns of dlx genes. dlx5a and dlx6a, known Edn1 targets, are downregulated in mef2ca mutant pharyngeal arch CNC. Surprisingly, dlx4b and dlx3b are oppositely affected in mef2ca mutants. dlx4b expression is abolished while the edn1-dependent dlx3b is ectopically expressed in more dorsal CNC. Together our results support a model in which CNC cells require mef2ca downstream of edn1 signaling for proper craniofacial development.

The crystal structure of the four-stranded DNA Holliday junction has now been determined in the presence and absence of junction binding proteins, with the extended open-X form of the junction seen in all protein complexes, but the more compact stacked-X structure observed in free DNA. The structures of the stacked-X junction were crystallized because of an unexpected sequence dependence on the stability of this structure. Inverted repeat sequences that contain the general motif NCC or ANC favor formation of stacked-X junctions, with the junction cross-over occurring between the first two positions of the trinucleotides. This review focuses on the sequence dependent structure of the stacked-X junction and how it may play a role in structural recognition by a class of dimeric junction resolving enzymes that themselves show no direct sequence recognition.

We have applied a comparative phylogenomic analysis to study the evolutionary relationships between GC content, CpG-dinucleotide content (CpGs), potential nuclear factor I (NFI) binding sites, and potential Z-DNA forming regions (ZDRs) as representative structural and functional GC-rich genomic elements. Our analysis indicates that CpG and NFI sites emerged with a general accretion of GC-rich sequences downstream of the eukaryotic transcription start site (TSS). Two distinct classes of ZDRs are observed at different locations proximal to the eukaryotic TSS. A robust CA/TG class of ZDRs was seen to emerge upstream of the TSS and independently of GC content, CpGs, and NFIs, whereas a second, weaker CG type appears to have evolved along with these downstream GC-rich elements. Taken together, the results provide a model for how GC-rich structural and functional eukaryotic markers emerge relative to each other, and indicate two distinct transition points for their occurrence: the first at the pro/eukaryotic boundary, and the second at or near the amniotic boundary.evolution ͉ genomic analysis ͉ Z-DNA

The single crystal structure of a DNA Holliday junction assembled from four unique sequences shows a structure that conforms to the general features of models derived from similar constructs in solution. The structure is a compact stacked-X form junction with two sets of stacked B-DNA type arms that coaxially stack to form semi-continuous duplexes interrupted only by the crossing of the junction. These semi-continuous helices are related by a right-handed rotation angle of 56.5°, which is nearly identical to the 60° angle in the solution model, but differ from the more shallow ~40° for previous crystal structures of symmetric junctions that self-assemble from single identical inverted-repeat sequences. This supports the model that the unique set of intramolecular interactions at the trinucleotide core of the crossing strands, which are not present in the current asymmetric junction, affect both the stability and geometry of the symmetric junctions. An unexpected result, however, is that a highly wobbled A·T base pair, which is ascribed here to a rare enol-tautomer form of the thymine, was observed at the end of a CCCC/GGGG sequence within the stacked B-DNA arms of this 1.9 Å resolution structure. We suggest that the junction itself is not responsible for this unusual conformation, but served as a vehicle to study this CG-rich sequence as a B-DNA duplex, mimicking the form that would be present in a replication complex. The existence of this unusual base lends credence to and defines a sequence context for the "rare tautomer hypothesis" as a mechanism for inducing transition mutations during DNA replication.The four-stranded DNA complex known as the Holliday junction is the central intermediate in homologous recombination and recombination mediated genetic mechanisms (1), including DNA repair and replication, resumption of stalled replication forks and viral genome integration (2-7). Although homologous recombination by definition involve symmetric sequences, asymmetric junctions can be assembled from four unique sequences to lock the position of DNA cross-over through base pair complementarity and, thereby, allow the structure and dynamics of junctions to be studied in solution (8) and in single-molecules (9, 10). Structural models derived from solution studies show that DNA junctions under physiological salt conditions adopt a compact "stacked-X" conformation (8) in which the arms pair and coaxially stack to form two near continuous duplexes that are related by an a 60° angle (the J twist (11)) across the junction cross-over (Fig. 1A).A number of single-crystal structures have now been reported of symmetric junctions that selfassembled from single inverted-repeat or near repeat sequences (12)(13)(14)(15)(16) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript intramolecular interactions that lock the tight U-turn of the cross-over to prevent migration of the junction in the crystals (13) and in solution (17). The crystal structures recapitulate the general features of the stacked-...