Mair E.A. Churchill

A purified mariner transposase is sufficient to mediate transposition in vitro

Mariners are a widespread and diverse family of animal transposons. Extremely similar mariners of the irritans subfamily are present in the genomes of three divergent insect host species, which strongly suggests that species-specific host factors are unnecessary for mobility. We tested this hypothesis by examining the activity of a purified transposase from one of these elements (Himar1) present in the horn fly, Haematobia irritans. Himar1 transposase was sufficient to reproduce transposition faithfully in an in vitro inter-plasmid transposition reaction. Further analyses showed that Himar1 transposase binds to the inverted terminal repeat sequences of its cognate transposon and mediates 5 and 3 cleavage of the element termini. Independence of species-specific host factors helps to explain why mariners have such a broad distribution and why they are capable of horizontal transfer between species.

Structural Basis and Specificity of Acyl-Homoserine Lactone Signal Production in Bacterial Quorum Sensing

Synthesis and detection of acyl-homoserine lactones (AHLs) enables many gram-negative bacteria to engage in quorum sensing, an intercellular signaling mechanism that activates differentiation to virulent and biofilm lifestyles. The AHL synthases catalyze acylation of S-adenosyl-L-methionine by acyl-acyl carrier protein and lactonization of the methionine moiety to give AHLs. The crystal structure of the AHL synthase, EsaI, determined at 1.8 A resolution, reveals a remarkable structural similarity to the N-acetyltransferases and defines a common phosphopantetheine binding fold as the catalytic core. Critical residues responsible for catalysis and acyl chain specificity have been identified from a modeled substrate complex and verified through functional analysis in vivo. A mechanism for the N-acylation of S-adenosyl-L-methionine by 3-oxo-hexanoyl-acyl carrier protein is proposed.

The structure of a chromosomal high mobility group protein-DNA complex reveals sequence-neutral mechanisms important for non-sequence-specific DNA recognition.

The high mobility group (HMG) chromosomal proteins, which are common to all eukaryotes, bind DNA in a non‐sequence‐specific fashion to promote chromatin function and gene regulation. They interact directly with nucleosomes and are believed to be modulators of chromatin structure. They are also important in V(D)J recombination and in activating a number of regulators of gene expression, including p53, Hox transcription factors and steroid hormone receptors, by increasing their affinity for DNA. The X‐ray crystal structure, at 2.2 Å resolution, of the HMG domain of the Drosophila melanogaster protein, HMG‐D, bound to DNA provides the first detailed view of a chromosomal HMG domain interacting with linear DNA and reveals the molecular basis of non‐sequence‐specific DNA recognition. Ser10 forms water‐mediated hydrogen bonds to DNA bases, and Val32 with Thr33 partially intercalates the DNA. These two ‘sequence‐neutral’ mechanisms of DNA binding substitute for base‐specific hydrogen bonds made by equivalent residues of the sequence‐specific HMG domain protein, lymphoid enhancer factor‐1. The use of multiple intercalations and water‐mediated DNA contacts may prove to be generally important mechanisms by which chromosomal proteins bind to DNA in the minor groove.

DNA chaperones: A solution to a persistence problem?

Andrew A. Travers, Sarbjit S. Ner, and Mair E. A. Churchill’ Medical Research Council Laboratory of Molecular Biology Cambridge CB2 2QH England *Department of Ceil and Structural Biology University of Illinois at Urbana-Champaign Urbana, Illinois 61601 In both the eukaryotic nucleus and the eubacterial nucleoid, nucleoprotein complexes establish and maintain the ordered packaging of the DNA genome. The paradigm for this mode of organization is the nucleosome core parti- cle in which the histone octamer wraps 145 bp of DNA in 1.6 left-handed superhelical turns, equivalent to an aver- age deflection of the double-helical axis by 47” for each turn of the duplex. This tight folding is in striking contrast with the solution properties of DNA, in which under physio- logical ionic conditions the DNA double helix possesses only limited flexibility. The experimentally determined per- sistence length (the length over which tangents to adjacent lengths tend to point in the same direction) of the polymer is 450-500 A or - 140 bp (reviewed by Hagerman, 1988). Such a fragment of DNA is not rigid, but can assume only a limited range of configurations and does not by itself attain a curvature comparable with that in the nucleosome core particle. Therefore, by trapping nearly two superheli- cal turns of DNA the nucleosome substantially reduces the end-to-end distance of such a DNA fragment (Figure 1). The required deformation of the double helix and the loss of entropy associated with the reduced conforma- tional freedom would be expected to incur a substantial energetic penalty on the formation of nucleosomes. The biological problem is how to build such condensed nucleoprotein structures in a chromosome. In vitro the as- sembly of the nucleosome core particle can be initiated by the histone H3/H4 tetramer that organizes 120 bp of DNA as a superhelix (Hansen et al., 1991; Hayes et al., 1991). How does this basic protein core trap the DNA in a superhelical configuration at a rate that will ensure rapid assembly? It is likely that after the initial contact with the tetramer, the DNA progressively wraps around the parti- cle. One way to facilitate this process is to prime the DNA for assembly into such a higher order complex, i.e., to prebend, pretwist, or both and to stabilize the DNA in a particular configuration that is able to transfer readily to the other proteins. Maintaining the DNA in such a configu- ration is analogous to the function of certain protein chap- erones, which stabilize a polypeptide in a conformation that is appropriate for subsequent assembly, but would be unstable without a chaperone. One such chaperone, nucleoplasmin, maintains histones in an accessible form for nucleosome assembly (Laskey et al., 1978). However, since the DNA undergoes a greater conformational change than the histones, it is likely that the optimal as-

Nonsequence-specific DNA recognition: a structural perspective.

Chromosomal proteins that form essential architectural components of chromatin bind and bend DNA with an intrinsic low degree of sequence preference. Comparisons made between two recently determined structures of high mobility group (HMG) protein-DNA complexes and other nonsequence-specific protein-DNA complexes reveal the structural basis of this important mode of DNA binding.

Structure of the Pseudomonas aeruginosa acyl‐homoserinelactone synthase LasI

The LasI/LasR quorum‐sensing system plays a pivotal role in virulence gene regulation of the opportunistic human pathogen, Pseudomonas aeruginosa. Here we report the crystal structure of the acyl‐homoserine lactone (AHL) synthase LasI that produces 3‐oxo‐C12‐AHL from the substrates 3‐oxo‐C12‐acyl‐carrier protein (acyl‐ACP) and S‐adenosyl‐L‐methionine. The LasI six‐stranded beta sheet platform, buttressed by three alpha helices, forms a V‐shaped substrate‐binding cleft that leads to a tunnel passing through the enzyme that can accommodate the acyl‐chain of acyl‐ACP. This tunnel places no apparent restriction on acyl‐chain length, in contrast to a restrictive hydrophobic pocket seen in the AHL‐synthase EsaI. Interactions of essential conserved N‐terminal residues, Arg23, Phe27 and Trp33, suggest that the N‐terminus forms an enclosed substrate‐binding pocket for S‐adenosyl‐L‐methionine. Analysis of AHL‐synthase surface residues identified a binding site for acyl‐ACP, a role that was supported by in vivo reporter assay analysis of the mutated residues, including Arg154 and Lys150. This structure and the novel explanation of AHL‐synthase acyl‐chain‐length selectivity promise to guide the design of Pseudomonas aeruginosa‐specific quorum‐sensing inhibitors as antibacterial agents.

The solution structure and dynamics of the DNA-binding domain of HMG-D from Drosophila melanogaster

BACKGROUNDnThe HMG-box is a conserved DNA-binding motif that has been identified in many high mobility group (HMG) proteins. HMG-D is a non-histone chromosomal protein from Drosophila melanogaster that is closely related to the mammalian HMG-box proteins HMG-1 and HMG-2. Previous structures determined for an HMG-box domain from rat and hamster exhibit the same global topology, but differ significantly in detail. It has been suggested that these differences may arise from hinge motions which allow the protein to adapt to the shape of its target DNA.nnnRESULTSnWe present the solution structure of HMG-D determined by NMR spectroscopy to an overall precision of 0.85 A root mean squared deviation (rmsd) for the backbone atoms. The protein consists of an extended amino-terminal region and three alpha-helices that fold into a characteristic L shape. The central core region of the molecule is highly stable and maintains an angle of approximately 80 degrees between the axes of helices 2 and 3. The backbone dynamics determined from 15N NMR relaxation measurements show a high correlation with the mean residue rmsd determined from the calculated structures.nnnCONCLUSIONSnThe structure determined for the HMG-box motif from HMG-D is essentially identical to the structure determined for the B-domain of mammalian HMG-1. Since these proteins have significantly different sequences our results indicate that the global fold and the mode of interaction with DNA are also likely to be conserved in all eukaryotes.

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

The high mobility group (HMG) protein HMG‐D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG‐D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T‐rich sequences. The TG motif constitutes a common core element in the binding sites of the well‐characterized sequence‐specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG‐D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG‐D can be considered an architecture‐specific protein. Finally, we show that HMG‐D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold‐associated region.

DNA binding of a non-sequence-specific HMG-D protein is entropy driven with a substantial non-electrostatic contribution

The thermal properties of two forms of the Drosophila melanogaster HMG-D protein, with and without its highly basic 26 residue C-terminal tail (D100 and D74) and the thermodynamics of their non-sequence-specific interaction with linear DNA duplexes were studied using scanning and titration microcalorimetry, spectropolarimetry, fluorescence anisotropy and FRET techniques at different temperatures and salt concentrations. It was shown that the C-terminal tail of D100 is unfolded at all temperatures, whilst the state of the globular part depends on temperature in a rather complex way, being completely folded only at temperatures close to 0 degrees C and unfolding with significant heat absorption at temperatures below those of the gross denaturational changes. The association constant and thus Gibbs energy of binding for D100 is much greater than for D74 but the enthalpies of their association are similar and are large and positive, i.e. DNA binding is a completely entropy-driven process. The positive entropy of association is due to release of counterions and dehydration upon forming the protein/DNA complex. Ionic strength variation showed that electrostatic interactions play an important but not exclusive role in the DNA binding of the globular part of this non-sequence-specific protein, whilst binding of the positively charged C-terminal tail of D100 is almost completely electrostatic in origin. This interaction with the negative charges of the DNA phosphate groups significantly enhances the DNA bending. An important feature of the non-sequence-specific association of these HMG boxes with DNA is that the binding enthalpy is significantly more positive than for the sequence-specific association of the HMG box from Sox-5, despite the fact that these proteins bend the DNA duplex to a similar extent. This difference shows that the enthalpy of dehydration of apolar groups at the HMG-D/DNA interface is not fully compensated by the energy of van der Waals interactions between these groups, i.e. the packing density at the interface must be lower than for the sequence-specific Sox-5 HMG box.

Modifying the helical structure of DNA by design: recruitment of an architecture-specific protein to an enforced DNA bend

BACKGROUNDnProteins can force DNA to adopt distorted helical structures that are rarely if ever observed in naked DNA. The ability to synthesize DNA that contains defined helical aberrations would offer a new avenue for exploring the structural and energetic plasticity of DNA. Here we report a strategy for the enforcement of non-canonical helical structures through disulfide cross-linking; this approach is exemplified by the design and synthesis of an oligonucleotide containing a pronounced bend.nnnRESULTSnA localized bend was site-specifically introduced into DNA by the formation of a disulfide cross-link between the 5 adenines of a 5-AATT-3 region in complementary strands of DNA. The DNA bend was characterized by high-resolution NMR structure determination of a cross-linked dodecamer and electrophoretic mobility assays on phased multimers, which together indicate that the cross-linked tetranucleotide induces a helical bend of approximately 30 degrees and a modest degree of unwinding. The enforced bend was found to stimulate dramatically the binding of an architecture-specific protein, HMG-D, to the DNA. DNase I foot-printing analysis revealed that the protein is recruited to the section of DNA that is bent.nnnCONCLUSIONSnThe present study reports a novel approach for the investigation of non-canonical DNA structures and their recognition by architecture-specific proteins. The mode of DNA bending induced by disulfide cross-linking resembles that observed in structures of protein-DNA complexes. The results reveal common elements in the DNA-binding mode employed by sequence-specific and architecture-specific HMG proteins.