Thomas D. Tullius

Hydroxyl radical footprinting: a high-resolution method for mapping protein-DNA contacts.

Publisher Summary This chapter discusses the utilization of the high resolution method “hydroxyl radical footprinting” for mapping protein–deoxyribonucleic acid (DNA) contacts. Hydroxyl radical cleaves DNA by abstracting a hydrogen atom from the deoxyribose sugars along the DNA backbone. As hydroxyl radical is exceedingly short lived and reactive and attacks sites on the surface of the DNA molecule, there is almost no sequence dependence or base dependence in the cleavage reaction. Every position along the backbone is cleaved nearly equally. This chemistry is used to determine the helical periodicity of a DNA-restriction fragment bound to an inorganic crystal, because hydroxyl radical could cut efficiently only the DNA backbone sugars that were directed away from the inorganic surface. The chapter illustrates that this same chemistry can be applied to produce the footprints of proteins bound to DNA as cutting of the DNA backbone by hydroxyl radical is blocked by bound protein. The chapter illustrates hydroxyl radical footprinting by its application to a well studied DNA–protein complex that of the bacteriophage λ repressor with the O R 1 operator DNA sequence. It also illustrates the way the reaction conditions for hydroxyl radical footprinting may be altered to accommodate other DNA-protein complexes.

Hydroxyl radical footprinting.

Publisher Summary This chapter provides an overview on hydroxyl radical footprinting. Hydroxyl radical footprinting of DNA has evolved over the last several years into a facile and powerful technique for studying DNA structure and complexes of DNA with other molecules. The chapter describes the chemistry and history behind these techniques. In a footprinting experiment, a strand of DNA is subjected to cleavage by some reagent, both in the presence and absence of a DNA-binding ligand. Comparison of the cleavage patterns yields information on the structure of the DNA-ligand complex. Footprinting can also be used to obtain thermodynamic information on DNA-ligand complexes through footprint titration experiments.

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. Topography-informed constrained regions correlated with functional noncoding elements, including enhancers, better than did regions identified solely on the basis of nucleotide sequence. These results support the idea that the molecular shape of DNA is under selection and can identify evolutionary history.

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. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal ‘arms’, and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.

The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc

BACKGROUND Integrase mediates a crucial step in the life cycle of the human immunodeficiency virus (HIV). The enzyme cleaves the viral DNA ends in a sequence-dependent manner and couples the newly generated hydroxyl groups to phosphates in the target DNA. Three domains have been identified in HIV integrase: an amino-terminal domain, a central catalytic core and a carboxy-terminal DNA-binding domain. The amino-terminal region is the only domain with unknown structure thus far. This domain, which is known to bind zinc, contains a HHCC motif that is conserved in retroviral integrases. Although the exact function of this domain is unknown, it is required for cleavage and integration. RESULTS The three-dimensional structure of the amino-terminal domain of HIV-2 integrase has been determined using two-dimensional and three-dimensional nuclear magnetic resonance data. We obtained 20 final structures, calculated using 693 nuclear Overhauser effects, which display a backbone root-mean square deviation versus the average of 0.25 A for the well defined region. The structure consists of three alpha helices and a helical turn. The zinc is coordinated with His 12 via the N epsilon 2 atom, with His16 via the N delta 1 atom and with the sulfur atoms of Cys40 and Cys43. The alpha helices form a three-helix bundle that is stabilized by this zinc-binding unit. The helical arrangement is similar to that found in the DNA-binding domains of the trp repressor, the prd paired domain and Tc3A transposase. CONCLUSION The amino-terminal domain of HIV-2 integrase has a remarkable hybrid structure combining features of a three-helix bundle fold with a zinc-binding HHCC motif. This structure shows no similarity with any of the known zinc-finger structures. The strictly conserved residues of the HHCC motif of retroviral integrases are involved in metal coordination, whereas many other well conserved hydrophobic residues are part of the protein core.

Using hydroxyl radical to probe DNA structure

Publisher Summary This chapter describes the application of the hydroxyl radical as a reagent for elucidating the structural details of DNA. The hydroxyl radical cleaves DNA by abstracting a hydrogen atom from a deoxyribose residue in the DNA backbone. Subsequently, the sugar breaks down, leaving a single-nucleoside gap with predominantly 5´- and 3´-phosphate ends. A small amount of 3´-phosphoglycolate is formed as well. This product migrates faster than the corresponding 3´-phosphate and can be seen when high percentage polyacrylamide gels are used to separate the products of hydroxyl radical cleavage of 5´-radiolabeled DNA. The examples presented in the chapter show that the hydroxyl radical cleavage experiment yields high resolution, reproducible structural data for DNA in solution. Adenine tracts in both a bent restriction fragment and a short oligonucleotide duplex have a characteristic pattern of hydroxyl radical cleavage that is related to the unusual conformation adopted by these tracts. The four strands of an immobile Holliday junction show distinctive hydroxyl radical protections that report on the crossover configuration of the molecule. Not only does this technique provide structural data at the nucleotide level, it can easily be performed by any laboratory that is experienced in the methods of molecular biology.

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. These maps reveal periodic structural signals in yeast and Drosophila genomic DNA sequences that are associated with positioned nucleosomes.

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. This method involves using the hydroxyl radical to cleave a nucleic acid molecule that is bound to a protein, followed by separating the cleavage products on a denaturing electrophoresis gel to identify the protein-binding sites on the nucleic acid molecule. We describe a protocol for hydroxyl radical footprinting of DNA–protein complexes, along with a troubleshooting guide, that allows researchers to obtain efficient cleavage of DNA in the presence and absence of proteins. This protocol can be completed in 2 d.

[56] Footprinting protein-DNA complexes with γ-rays

Publisher Summary This chapter discusses the footprinting of protein–DNA complexes using γ rays. It presents an analog of the method of hydroxyl radical footprinting. The high-resolution footprints is duplicated of this technique, while substituting γ-radiation for the chemical reagents used heretofore. γ-Radiation has been shown to mediate DNA cleavage with no apparent base or sequence specificity. The primary cutting reagent, as in the analogous chemical reagent system, is thought to be the hydroxyl radical. This method requires no addition of reagents to the protein-DNA binding solution. The technique can be used to carry out footprinting in protein–DNA binding solutions that are not conducive to the use of the [Fe(II)EDTA] 2-reagent system, such as those that contain moderate amounts of glycerol or those that cannot tolerate the addition of one of the reagent components, such as hydrogen peroxide. Morever, the ability of γ-radiation to penetrate matter suggests that extension of the technique to in vivo systems might be possible. Such experiments could be performed on a model system constructed on a plasmid or could be applied to interactions of proteins with chromosomal DNA when used in combination with an appropriate detection scheme. An in vivo application would offer the opportunity of determining high-resolution footprints of proteins bound to DNA inside cells.

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. It is becoming clear that DNA shape is important to biological function, and therefore will be subject to evolutionary constraint.