Structure and operation of the DNA-translocating type I DNA restriction enzymes.

Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.

Large multimeric assemblies of nucleosome assembly protein and histones revealed by small-angle X-ray scattering and electron microscopy.

The nucleosome assembly protein (NAP) family represents a key group of histone chaperones that are essential for cell viability. Several x-ray structures of NAP1 dimers are available; however, there are currently no structures of this ubiquitous chaperone in complex with histones. We have characterized NAP1 from Xenopus laevis and reveal that it forms discrete multimers with histones H2A/H2B and H3/H4 at a stoichiometry of one NAP dimer to one histone fold dimer. These complexes have been characterized by size exclusion chromatography, analytical ultracentrifugation, multiangle laser light scattering, and small-angle x-ray scattering to reveal their oligomeric assembly states in solution. By employing single-particle cryo-electron microscopy, we visualized these complexes for the first time and show that they form heterogeneous ring-like structures, potentially acting as large scaffolds for histone assembly and exchange.

A rapid purification procedure for the HsdM protein of EcoR124I and biophysical characterization of the purified protein.

Type I restriction-modification (R-M) systems are comprised of two multi-subunit enzymes with complementary functions: the methyltransferase (~160 kDa), responsible for methylation of DNA, and the restriction endonuclease (~400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits, including HsdM. Characterisation of either enzyme first requires the expression and purification of its constituent subunits, before reconstitution of the multisubunit complex. Previously, purification of the HsdM protein had proved problematic, due to the length of time required for the purification and its susceptibility to degradation. A new protocol was therefore developed to decrease the length of time required to purify the HsdM protein and thus prevent degradation. Finally, we show that the HsdM subunit exhibits a concentration dependent monomer-dimer equilibrium.

Predicting the effects of basepair mutations in DNA-protein complexes by thermodynamic integration.

Thermodynamically rigorous free energy methods in principle allow the exact computation of binding free energies in biological systems. Here, we use thermodynamic integration together with molecular dynamics simulations of a DNA-protein complex to compute relative binding free energies of a series of mutants of a protein-binding DNA operator sequence. A guanine-cytosine basepair that interacts strongly with the DNA-binding protein is mutated into adenine-thymine, cytosine-guanine, and thymine-adenine. It is shown that basepair mutations can be performed using a conservative protocol that gives error estimates of ∼10% of the change in free energy of binding. Despite the high CPU-time requirements, this work opens the exciting opportunity of being able to perform basepair scans to investigate protein-DNA binding specificity in great detail computationally.

Structural analysis of the genetic switch that regulates the expression of restriction-modification genes.

Controller (C) proteins regulate the timing of the expression of restriction and modification (R-M) genes through a combination of positive and negative feedback circuits. A single dimer bound to the operator switches on transcription of the C-gene and the endonuclease gene; at higher concentrations, a second dimer bound adjacently switches off these genes. Here we report the first structure of a C protein-DNA operator complex, consisting of two C protein dimers bound to the native 35 bp operator sequence of the R-M system Esp1396I. The structure reveals a role for both direct and indirect DNA sequence recognition. The structure of the DNA in the complex is highly distorted, with severe compression of the minor groove resulting in a 50 degrees bend within each operator site, together with a large expansion of the major groove in the centre of the DNA sequence. Cooperative binding between dimers governs the concentration-dependent activation-repression switch and arises, in part, from the interaction of Glu25 and Arg35 side chains at the dimer-dimer interface. Competition between Arg35 and an equivalent residue of the sigma(70) subunit of RNA polymerase for the Glu25 site underpins the switch from activation to repression of the endonuclease gene.

A competition assay for DNA binding using the fluorescent probe ANS.

Fluorescence spectroscopy is a technique frequently employed to study protein-nucleic acid interactions. Often, the intrinsic fluorescence emission spectrum of tryptophan residues in a nucleic-acid-binding protein is strongly perturbed upon interaction with a target DNA or RNA. These spectral changes can then be exploited in order to construct binding isotherms and the extract equilibrium association constant together with the stoichiometry of an interaction. However, when a protein contains many tryptophan residues that are not located in the proximity of the nucleic-acid-binding site, changes in the fluorescence emission spectrum may not be apparent or the magnitude too small to be useful. Here, we make use of an extrinsic fluorescence probe, the environmentally sensitive fluorophore 1-anilinonaphthalene-8-sulphonic acid (1,8-ANS). Displacement by DNA of 1,8-ANS molecules from the nucleic-acid-binding site of the Type I modification methylase EcoR124I results in red shifting and an intensity decrease of the 1,8-ANS fluorescence emission spectrum. These spectral changes have been used to investigate the interaction of EcoR124I with DNA target recognition sequences.

Circular dichroism for the analysis of protein-DNA interactions.

Circular dichroism (CD) is a well-established technique for the analysis of both protein and DNA structure. The analysis of protein-nucleic acid complexes presents greater challenges, but at wavelengths above 250 nm, the circular dichroism signal from the DNA predominates. Examples are given of the use of CD to examine structural changes to DNA induced by protein binding.

Fluorescence spectroscopy and anisotropy in the analysis of DNA-protein interactions.

Fluorescence spectroscopy can be used as a sensitive non-destructive technique for the characterisation of protein-DNA interactions. A comparison of the intrinsic emission spectra obtained for a protein-DNA complex and for free protein can be informative about the environment of tryptophan and tyrosine residues in the two states. Often there is quenching of the fluorescence intensity of an intrinsic emission spectrum and/or a shift in the wavelength maximum on protein binding to DNA. A step-by-step protocol describes the determination of a DNA-binding curve by measurement of the quenching of the intrinsic protein fluorescence.Fluorescence anisotropy can also be used to obtain a DNA-binding curve if the molecular size of the protein-DNA complex is sufficiently different from the free fluorescing component. Typically an extrinsic fluorophore attached to one or both 5' ends of single-stranded or duplex DNA is used, for this increases the sensitivity of measurement.Fitting of the binding curves, assuming a model, can often yield the stoichiometry and association constant of the interaction. The approach is illustrated using the interaction of the DNA-binding domains (HMG boxes) of mouse Sox-5 and mammalian HMGB1 with short DNA duplexes.

Alternative splicing determines the interaction of SMRT isoforms with nuclear receptor-DNA complexes.

Signalling by small molecules, such as retinoic acid, is mediated by heterodimers comprising a class II nuclear receptor and an RXR (retinoid X receptor) subunit. The receptors bind to DNA response elements and act as ligand-dependent transcription factors, but, in the absence of signal, the receptors bind the co-repressors SMRT [silencing mediator for RAR (retinoic acid receptor) and TR (thyroid hormone receptor)] and NCoR (nuclear receptor co-repressor) and repress gene expression. Alternative splicing of the SMRT transcript in mammals generates six isoforms containing 1, 2 or 3 CoRNR (co-repressor for nuclear receptor) box motifs which are responsible for the interactions with nuclear receptors. We show that human cell lines express all six SMRT isoforms and then determine the binding affinity of mouse SMRT isoforms for RAR/RXR and three additional class II nuclear receptor-DNA complexes. This approach demonstrates the importance of the full complement of CoRNR boxes within each SMRT protein, rather than the identity of individual CoRNR boxes, in directing the interaction of SMRT with nuclear receptors. Each class of SMRT isoform displays a distinct feature, as the 1-box isoform discriminates between DNA response elements, the 2-box isoforms promote high-affinity binding to TR complexes and the 3-box isoforms show differential binding to nuclear receptors. Consequently, the differential deployment of SMRT isoforms observed in vivo could significantly expand the regulatory capacity of nuclear receptor signalling.

Structural and functional characterisation of the DNA binding domain of the Aspergillus nidulans gene regulatory protein AreA.

The 876-aa protein AreA regulates the expression of numerous genes involved in nitrogen metabolism in Aspergillus nidulans, and interacts with GATA sequences upstream of the relevant genes. We have carried out limited proteolysis of the C-terminal domain of the AreA protein in order to identify possible structural domains within the protein. A stable 156-amino-acid fragment was identified that contained the zinc finger region, and this sequence was cloned and expressed in E. coli. Fluorescence spectroscopy of the purified protein showed that the proteolytic domain was folded and could be denatured by high concentrations of urea (approximately 4 M), exhibiting a sharp transition. Fluorescence spectroscopy was also used to monitor binding to a DNA duplex containing the AreA recognition site, demonstrating tight binding of the domain to its DNA recognition sequence. The DNA binding affinity of the domain is comparable with that of the native AreA protein and much higher than that of the minimal zinc finger region of AreA.

Structural and functional analysis of the engineered type I DNA methyltransferase EcoR124I(NT).

The Type I R-M system EcoR124I is encoded by three genes. HsdM is responsible for modification (DNA methylation), HsdS for DNA sequence specificity and HsdR for restriction endonuclease activity. The trimeric methyltransferase (M(2)S) recognises the asymmetric sequence (GAAN(6)RTCG). An engineered R-M system, denoted EcoR124I(NT), has two copies of the N-terminal domain of the HsdS subunit of EcoR124I, instead of a single S subunit with two domains, and recognises the symmetrical sequence GAAN(7)TTC. We investigate the methyltransferase activity of EcoR124I(NT), characterise the enzyme and its subunits by analytical ultracentrifugation and obtain low-resolution structural models from small-angle neutron scattering experiments using contrast variation and selective deuteration of subunits.

HsdR subunit of the type I restriction-modification enzyme EcoR124I: biophysical characterisation and structural modelling.

Type I restriction-modification (RM) systems are large, multifunctional enzymes composed of three different subunits. HsdS and HsdM form a complex in which HsdS recognizes the target DNA sequence, and HsdM carries out methylation of adenosine residues. The HsdR subunit, when associated with the HsdS-HsdM complex, translocates DNA in an ATP-dependent process and cleaves unmethylated DNA at a distance of several thousand base-pairs from the recognition site. The molecular mechanism by which these enzymes translocate the DNA is not fully understood, in part because of the absence of crystal structures. To date, crystal structures have been determined for the individual HsdS and HsdM subunits and models have been built for the HsdM-HsdS complex with the DNA. However, no structure is available for the HsdR subunit. In this work, the gene coding for the HsdR subunit of EcoR124I was re-sequenced, which showed that there was an error in the published sequence. This changed the position of the stop codon and altered the last 17 amino acid residues of the protein sequence. An improved purification procedure was developed to enable HsdR to be purified efficiently for biophysical and structural analysis. Analytical ultracentrifugation shows that HsdR is monomeric in solution, and the frictional ratio of 1.21 indicates that the subunit is globular and fairly compact. Small angle neutron-scattering of the HsdR subunit indicates a radius of gyration of 3.4 nm and a maximum dimension of 10 nm. We constructed a model of the HsdR using protein fold-recognition and homology modelling to model individual domains, and small-angle neutron scattering data as restraints to combine them into a single molecule. The model reveals an ellipsoidal shape of the enzymatic core comprising the N-terminal and central domains, and suggests conformational heterogeneity of the C-terminal region implicated in binding of HsdR to the HsdS-HsdM complex.