Experimental maps of DNA structure at nucleotide resolution distinguish intrinsic from protein-induced DNA deformations.

Recognition of DNA by proteins depends on DNA sequence and structure. Often unanswered is whether the structure of naked DNA persists in a protein-DNA complex, or whether protein binding changes DNA shape. While X-ray structures of protein-DNA complexes are numerous, the structure of naked cognate DNA is seldom available experimentally.

GBshape: a genome browser database for DNA shape annotations.

Many regulatory mechanisms require a high degree of specificity in protein-DNA binding. Nucleotide sequence does not provide an answer to the question of why a protein binds only to a small subset of the many putative binding sites in the genome that share the same core motif. Whereas higher-order effects, such as chromatin accessibility, cooperativity and cofactors, have been described, DNA shape recently gained attention as another feature that fine-tunes the DNA binding specificities of some transcription factor families.

Chemical probing of RNA with the hydroxyl radical at single-atom resolution.

While hydroxyl radical cleavage is widely used to map RNA tertiary structure, lack of mechanistic understanding of strand break formation limits the degree of structural insight that can be obtained from this experiment. Here, we determine how individual ribose hydrogens of sarcin/ricin loop RNA participate in strand cleavage. We find that substituting deuterium for hydrogen at a ribose 5'-carbon produces a kinetic isotope effect on cleavage; the major cleavage product is an RNA strand terminated by a 5'-aldehyde.

Comparative analysis of metazoan chromatin organization.

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization.

A map of minor groove shape and electrostatic potential from hydroxyl radical cleavage patterns of DNA.

DNA shape variation and the associated variation in minor groove electrostatic potential are widely exploited by proteins for DNA recognition. Here we show that the hydroxyl radical cleavage pattern is a quantitative measure of DNA backbone solvent accessibility, minor groove width, and minor groove electrostatic potential, at single nucleotide resolution. We introduce maps of DNA shape and electrostatic potential as tools for understanding how proteins recognize binding sites in a genome.

A computational method to search for DNA structural motifs in functional genomic elements.

The rapidly increasing availability of DNA sequence data from modern high-throughput experimental techniques has created the need for computational algorithms to aid in motif discovery in genomic DNA. Such algorithms are typically used to find a statistical representation of the nucleotide sequence of the target site of a DNA-binding protein within a collection of DNA sequences that are thought to contain segments to which the protein is bound. A major assumption of these algorithms is that the protein recognizes the primary order of nucleotides in the sequence.

DNA shape, genetic codes, and evolution.

Although the three-letter genetic code that maps nucleotide sequence to protein sequence is well known, there must exist other codes that are embedded in the human genome. Recent work points to sequence-dependent variation in DNA shape as one mechanism by which regulatory and other information could be encoded in DNA. Recent advances include the discovery of shape-dependent recognition of DNA that depends on minor groove width and electrostatics, the existence of overlapping codes in protein-coding regions of the genome, and evolutionary selection for compensatory changes in nucleotide composition that facilitate nucleosome occupancy.

Local DNA topography correlates with functional noncoding regions of the human genome.

The three-dimensional molecular structure of DNA, specifically the shape of the backbone and grooves of genomic DNA, can be dramatically affected by nucleotide changes, which can cause differences in protein-binding affinity and phenotype. We developed an algorithm to measure constraint on the basis of similarity of DNA topography among multiple species, using hydroxyl radical cleavage patterns to interrogate the solvent-accessible surface area of DNA. This algorithm found that 12% of bases in the human genome are evolutionarily constrained-double the number detected by nucleotide sequence-based algorithms.

Footprinting protein-DNA complexes using the hydroxyl radical.

Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA-protein complexes. The high reactivity and lack of base specificity of the hydroxyl radical makes it an excellent probe for high-resolution footprinting of DNA-protein complexes; this technique can provide structural detail that is not achievable using DNase I footprinting. Hydroxyl radical footprinting experiments can be carried out using readily available and inexpensive reagents and lab equipment.

Probing DNA structure with hydroxyl radicals.

The hydroxyl radical is a useful probe for studying the shape of the surface of a DNA molecule. Using this technique, fine details of DNA structure can potentially be revealed. This unit describes how to use the hydroxyl radical to generate a random cleavage pattern at the surface of the molecule, separate the broken DNA strands by polyacrylamide gel electrophoresis, and analyze the cleavage pattern to give an image of the surface of the molecule.

The relationship between fine scale DNA structure, GC content, and functional elements in 1% of the human genome.

GC content has been shown to be an important aspect of human genomic function. Extending beyond the scope of GC content alone, there is a class of regions in the genome that have especially high GC content and are enriched for the CG dinucleotide--called CpG islands. CpG islands have been linked to biologically functional genomic elements.

Effects of discontinuities in the DNA template on abortive initiation and promoter escape by Escherichia coli RNA polymerase.

Using singly gapped or nicked templates containing the T7A1 promoter, we have measured several kinetic parameters related to the process of transcription initiation by Escherichia coli RNA polymerase, confirming and extending previous results using a population of randomly gapped templates. A reduced probability of transcript abortion at RNA lengths of 6 and 7 nucleotides and a lower ratio of abortive to productive initiation events was observed for some discontinuous templates, consistent with models attributing abortive initiation to the accumulation of strain in the initiating complex. The effect of DNA discontinuity on abortion of shorter RNA transcripts (2-3 nucleotides) was less pronounced; abortion at these short chain lengths may primarily be attributed to the low stability of the RNA-DNA hybrid.

Construction of a genome-scale structural map at single-nucleotide resolution.

Few methods are available for mapping the local structure of DNA throughout a genome. The hydroxyl radical cleavage pattern is a measure of the local variation in solvent-accessible surface area of duplex DNA, and thus provides information on the local shape and structure of DNA. We report the construction of a relational database, ORChID (OH Radical Cleavage Intensity Database), that contains extensive hydroxyl radical cleavage data produced from two DNA libraries.

Detection of DNA structural motifs in functional genomic elements.

The completion of the human genome project has fueled the search for regulatory elements by a variety of different approaches. Many successful analyses have focused on examining primary DNA sequence and/or chromatin structure. However, it has been difficult to detect common sequence motifs within the feature of chromatin structure most closely associated with regulatory elements, DNase I hypersensitive sites (DHSs).

Platinum anticancer drug damage enforces a particular rotational setting of DNA in nucleosomes.

We constructed two site-specifically modified nucleosomes containing an intrastrand cis-{Pt(NH3)2}2+ 1,3-d(GpTpG) cross-link, similar to one formed by the anticancer drugs carboplatin and cisplatin on DNA, and investigated their structures by hydroxyl radical footprinting and exonuclease III digestion. Hydroxyl radical footprinting demonstrated that the presence of the platinum cross-link selects out a specific rotational setting of DNA on the histone octamer core in each of two reconstituted nucleosomes in which the platinum positions differ by half a DNA helical turn. The {Pt(NH3)2}2+ cross-link is situated in a structurally similar location, with the undamaged strand projecting outward, forcing the DNA to adopt opposite rotational settings in its wrapping around the histone octamer in the two nucleosomes.

Mapping nucleic acid structure by hydroxyl radical cleavage.

Hydroxyl radical footprinting is a widely used method for following the folding of RNA molecules in solution. This method has the unique ability to provide experimental information on the solvent accessibility of each nucleotide in an RNA molecule, so that the folding of all domains of the RNA species can be followed simultaneously at single-nucleotide resolution. In recent work, hydroxyl radical footprinting has been used, often in combination with other global measures of structure, to work out detailed folding pathways and three-dimensional structures for increasingly large and complicated RNA molecules.

Gapped DNA is anisotropically bent.

Ionizing radiation damages DNA in several ways, including through formation of a single-nucleoside gap in one DNA strand. We have developed a two-dimensional gel electrophoresis method to investigate the effect of a strand gap on DNA structure. We generate a library of gapped DNA molecules by treating a DNA restriction fragment with the hydroxyl radical, generated by the reaction of Fe(II) EDTA with hydrogen peroxide.

Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm.

The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different.

A general synthesis of specifically deuterated nucleotides for studies of DNA and RNA.

An efficient procedure is described for the preparation of ribonucleotides and deoxyribonucleotides with deuterium incorporated at the 1', 4', or 5' position. Three intermediates-[1-2H]-D-ribose, [4-2H]-D-ribose, and [5-2H(2)]-D-ribose-were prepared by chemical synthesis and subsequently converted to ribonucleotides and deoxyribonucleotides via enzymatic reactions. Milligram quantities of the desired products were obtained with an average deuterium content of 96+/-1%.