Mechanism Of DNA Recognition Research Articles

Real-Time Observation of DNA Recognition by the RNA-Guided Endonuclease Cas9 using Single-Molecule FRET

The RNA-guided endonuclease Cas9 targets foreign DNA for degradation as part of an adaptive immune system in bacteria mediated by clustered regularly interspaced short palindromic repeats (CRISPR). Due in part to the ease of programmability, Cas9 is now being widely used in various organisms for site-specific genomic editing, genome-wide knockout screens, and transcriptional activation and repression. This system brings together three major classes of biopolymers (DNA, RNA and protein), but the specificity of different components for each other remains poorly understood. Perhaps most important, Cas9 has been shown to bind and cleave genomic DNA sequences with varying degrees of mismatches to the guide RNA, leading to potentially deleterious off-target effects. Previous methods to investigate the tolerability of mismatches have largely employed experimental methods that use Cas9-induced DNA cleavage as a readout of specificity and not direct binding. Recent ChIP-seq experiments suggest that off-target binding occurs at sites with complementarity in just a short 5-bp seed region, but the use of crosslinking obscures kinetic information and can potentially introduce artifacts. Here, we have used single-molecule FRET together with bulk biochemical assays to directly observe interactions between Cas9-RNA and DNA targets of varying sequence complementarity in real time. Using multiple distinct labeling geometries that report on both initial association and subsequent DNA unwinding, we develop a kinetic framework for the RNA-guided DNA interrogation process and provide insight into conformational changes that are required for target cleavage. Our findings resolve outstanding questions on the mechanism of DNA recognition by Cas9 and will facilitate improvements in genome engineering applications.

In Vitro Reconstitution and Crystallization of Cas9 Endonuclease Bound to a Guide RNA and a DNA Target.

The programmable RNA-guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas9 is the basis of genome editing applications in numerous model organisms and cell types. In a binary complex with a dual crRNA:tracrRNA guide or single-molecule guide RNA, Cas9 targets double-stranded DNAs harboring sequences complementary to a 20-nucleotide segment in the guide RNA. Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-guided DNA recognition. Here, we provide protocols for electrophoretic mobility shift and fluorescence-detection size exclusion chromatography assays used to probe DNA binding by Cas9 that allowed us to reconstitute and crystallize the enzyme in a ternary complex with a guide RNA and a bona fide target DNA. The procedures can be used for further mechanistic investigations of the Cas9 endonuclease family and are potentially applicable to other multicomponent protein-nucleic acid complexes.

Extreme tolerance and developmental buffering of UV-C induced DNA damage in embryos of the annual killifish Austrofundulus limnaeus.

Free-living aquatic embryos are often at risk of exposure to ultraviolet radiation (UV-R). Successful completion of embryonic development depends on efficient removal of DNA lesions, and thus many aquatic embryos have mechanisms to reverse DNA lesions induced by UV-R. However, little is known of how embryos that are able to enter embryonic dormancy may respond to UV-R exposure and subsequent DNA damage. Embryos of the annual killifish Austrofundulus limnaeus are unique among vertebrates because their normal embryonic development includes (1) a complete dispersion of embryonic blastomeres prior to formation of the definitive embryonic axis, and (2) entry into a state of metabolic depression and developmental arrest termed diapause. Here, we show that developing and diapausing embryos of A. limnaeus have exceptional tolerance of UV-C radiation and can successfully complete embryonic development after receiving substantial doses of UV-C, especially if allowed to recover in full-spectrum light. Recovery in full-spectrum light permits efficient removal of the most common type of DNA lesion induced by UV-R: cyclobutane pyrimidine dimers. Interestingly, whole-mount embryo TUNEL assays suggest that apoptosis may not be a major contributor to cell death in embryos UV-C irradiated during dispersion/reaggregation or diapause. We also observed embryo mortality to be significantly delayed by several weeks in diapausing embryos irradiated and allowed to recover in the dark. These atypical responses to UV-R induced DNA damage may be due to the unique annual killifish life history and provide insight into DNA damage repair and recognition mechanisms during embryonic dormancy.

Chemical display of pyrimidine bases flipped out by modification-dependent restriction endonucleases of MspJI and PvuRts1I families.

The epigenetic DNA modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in eukaryotes are recognized either in the context of double-stranded DNA (e.g., by the methyl-CpG binding domain of MeCP2), or in the flipped-out state (e.g., by the SRA domain of UHRF1). The SRA-like domains and the base-flipping mechanism for 5(h)mC recognition are also shared by the recently discovered prokaryotic modification-dependent endonucleases of the MspJI and PvuRts1I families. Since the mechanism of modified cytosine recognition by many potential eukaryotic and prokaryotic 5(h)mC “readers” is still unknown, a fast solution based method for the detection of extrahelical 5(h)mC would be very useful. In the present study we tested base-flipping by MspJI- and PvuRts1I-like restriction enzymes using several solution-based methods, including fluorescence measurements of the cytosine analog pyrrolocytosine and chemical modification of extrahelical pyrimidines with chloroacetaldehyde and KMnO4. We find that only KMnO4 proved an efficient probe for the positive display of flipped out pyrimidines, albeit the method required either non-physiological pH (4.3) or a substitution of the target cytosine with thymine. Our results imply that DNA recognition mechanism of 5(h)mC binding proteins should be tested using a combination of all available methods, as the lack of a positive signal in some assays does not exclude the base flipping mechanism.

DNA binding and dimerization specificities of Basic Zipper transcription factors

The ability of basic zipper transcription factors to form homo- or heterodimers provides a paradigm for combinatorial control of eukaryotic gene expression. In a first study, we clarified the specificity of the MIcrophthalmia-associated Transcription Factor [1]. To achieve this, we solved three crystal structures: two structures of MITF in complex with DNA duplexes encompassing two different target motifs (E-box and M-box) and one APO-structure. We then analyzed interactions between these DNA elements and several MITF mutants with documented mice phenotypes, using complementary techniques. The comparison of these experiments together with available biological data reveals the particular mechanism of DNA recognition by MITF. Moreover we demonstrated how a shift in the leucine zipper register limits the choice of the homotypic dimerization partner among the other b-HLH-Zip transcription factors. In a second study, we wondered how facultative dimerization results in alternative DNA-binding repertoires on distinct regulatory elements [2]. In this respect, the hematopoietic b-Zip transcription factor MafB, is a good model, since it has the ability to form homo- and heterodimers with a few other transcription factors. We first determined two high-resolution structures of MafB as a homodimer and as a heterodimer with c-Fos bound to variants of the Maf-recognition element (MARE). The two structures revealed several unexpected and specific coiled coil interactions. Based on these findings, we have engineered two MafB mutants with opposite dimerization preferences. One of them indeed showed a strong preference for MafB/c-Fos heterodimerization. In addition this variant enabled a selection of heterodimer- favoring over homodimer-specific MARE variants, demonstrating that protein/protein and protein/DNA interactions are interconnected. Our data provide a new concept for transcription factor design to selectively activate dimer-specific pathways and binding repertoires.

Target Recognition and Degradation by an Adaptive Bacterial Immune System

Prokaryotes use the CRISPR adaptive immune system to defend against invasive genetic elements. In Type I systems, short pieces of acquired DNA are transcribed into CRISPR RNAs (crRNAs) and loaded into the multi-subunit complex Cascade for target identification. The effector nuclease Cas3 is then recruited for target DNA degradation. Here we present single-particle negative stain and cryo-electron microscopy reconstructions of the RNA-guided surveillance complex from the E. coli bacterial immune system. These structures, along with biochemical studies, provide insight into the mechanisms of initial DNA recognition, effector nuclease recruitment, and target degradation, which are essential steps of CRISPR-based immunity.

Structural mechanism of DNA recognition by the p202 HINa domain: insights into the inhibition of Aim2-mediated inflammatory signalling.

The HIN-200 family of proteins play significant roles in inflammation-related processes. Among them, AIM2 (absent in melanoma 2) and IFI16 (γ-interferon-inducible protein 16) recognize double-stranded DNA to initiate inflammatory responses. In contrast, p202, a mouse interferon-inducible protein containing two HIN domains (HINa and HINb), has been reported to inhibit Aim2-mediated inflammatory signalling in mouse. To understand the inhibitory mechanism, the crystal structure of the p202 HINa domain in complex with a 20 bp DNA was determined, in which p202 HINa nonspecifically recognizes both strands of DNA through electrostatic attraction. The p202 HINa domain binds DNA more tightly than does AIM2 HIN, and the DNA-binding mode of p202 HINa is different from that of the AIM2 HIN and IFI16 HINb domains. These results, together with the reported data on p202 HINb, lead to an interaction model for full-length p202 and dsDNA which provides a conceivable mechanism for the negative regulation of Aim2 inflammasome activation by p202.

Type III restriction-modification enzymes: a historical perspective.

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction–modification (R–M) systems are classified into four groups. Type III R–M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25–27 bp downstream of one of the recognition sites). Like the Type I R–M enzymes, Type III R–M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R–M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R–M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

Investigation of DNA sequence recognition by a streptomycete MarR family transcriptional regulator through surface plasmon resonance and X-ray crystallography

Consistent with their complex lifestyles and rich secondary metabolite profiles, the genomes of streptomycetes encode a plethora of transcription factors, the vast majority of which are uncharacterized. Herein, we use Surface Plasmon Resonance (SPR) to identify and delineate putative operator sites for SCO3205, a MarR family transcriptional regulator from Streptomyces coelicolor that is well represented in sequenced actinomycete genomes. In particular, we use a novel SPR footprinting approach that exploits indirect ligand capture to vastly extend the lifetime of a standard streptavidin SPR chip. We define two operator sites upstream of sco3205 and a pseudopalindromic consensus sequence derived from these enables further potential operator sites to be identified in the S. coelicolor genome. We evaluate each of these through SPR and test the importance of the conserved bases within the consensus sequence. Informed by these results, we determine the crystal structure of a SCO3205-DNA complex at 2.8 Å resolution, enabling molecular level rationalization of the SPR data. Taken together, our observations support a DNA recognition mechanism involving both direct and indirect sequence readout.

Mechanism of Origin DNA Recognition and Assembly of an Initiator-Helicase Complex by SV40 Large Tumor Antigen

The DNA tumor virus Simian virus 40 (SV40) is a model system for studying eukaryotic replication. SV40 large tumor antigen (LTag) is the initiator/helicase that is essential for genome replication. LTag recognizes and assembles at the viral replication origin. We determined the structure of two multidomain LTag subunits bound to origin DNA. The structure reveals that the origin binding domains (OBDs) and Zn and AAA+ domains are involved in origin recognition and assembly. Notably, the OBDs recognize the origin in an unexpected manner. The histidine residues of the AAA+ domains insert into a narrow minor groove region with enhanced negative electrostatic potential. Computational analysis indicates that this region is intrinsically narrow, demonstrating the role of DNA shape readout in origin recognition. Our results provide important insights into the assembly of the LTag initiator/helicase at the replication origin and suggest that histidine contacts with the minor groove serve as a mechanism of DNA shape readout.

Recognition of specific and nonspecific DNA by human lactoferrin

The general principles of recognition of nucleic acids by proteins are among the most exciting problems of molecular biology. Human lactoferrin (LF) is a remarkable protein possessing many independent biological functions, including interaction with DNA. In human milk, LF is a major DNase featuring two DNA-binding sites with different affinities for DNA. The mechanism of DNA recognition by LF was studied here for the first time. Electrophoretic mobility shift assay and fluorescence measurements were used to probe for interactions of the high-affinity DNA-binding site of LF with a series of model-specific and nonspecific DNA ligands, and the structural determinants of DNA recognition by LF were characterized quantitatively. The minimal ligands for this binding site were orthophosphate (K(i) = 5 mM), deoxyribose 5'-phosphate (K(i) = 3 mM), and different dNMPs (K(i) = 0.56-1.6 mM). LF interacted additionally with 9-12 nucleotides or nucleotide pairs of single- and double-stranded ribo- and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleoside phosphate groups. Such nonspecific interactions of LF with noncognate single- and double-stranded d(pN)(10) provided ~6 to ~7.5 orders of magnitude of the enzyme affinity for any DNA. This corresponds to the Gibbs free energy of binding (ΔG(0)) of -8.5 to -10.0 kcal/mol. Formation of specific contacts between the LF and its cognate DNA results in an increase of the DNA affinity for the enzyme by approximately 1 order of magnitude (K(d) = 10 nM; ΔG(0) ≈ -11.1 kcal/mol). A general function for the LF affinity for nonspecific d(pN)(n) of different sequences and lengths was obtained, giving the K(d) values comparable with the experimentally measured ones. A thermodynamic model was constructed to describe the interactions of LF with DNA.

Deformation of a P53 Response Element Revealed by Site-Directed Spin Labeling

The tumor suppressor protein p53 is called the “guardian of the genome” and has been implicated in a growing number of biological processes, including cell cycle arrest, senescence, apoptosis, autophagy, metabolism, and aging. As a transcription factor, p53 regulates a large number of signaling pathways by interacting with a diverse set of DNAs, called p53 response elements (REs), which are composed of two decameric consensus half-sites separated by a spacer. It has been proposed that DNA conformation changes upon p53 binding, which may vary between different REs, play important roles in p53-DNA recognition. However, details on such DNA conformational changes are limited, as currently there is no high-resolution structure of unbound RE, although a number of crystal structures of tetrameric p53 fragments bound to artificial or naturally occurring REs have been reported. Here site-directed spin labeling is used to probe solution conformations of an RE regulating protein p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation. Using a nucleotide-independent nitroxide probe and Double-Electron-Electron Resonance spectroscopy, nanometer distances between various DNA sites were measured in unbound p21RE as well as in that complexed to the p53 DNA binding domain. Models of the unbound p21RE, which were selected using the measured distances from a large pool generated by Monte-Carlo simulations, reveal major conformational changes at the half-site interface as compared to the bound DNA reported in a crystal structure. These results shed light on the mechanism of DNA recognition by p53.

3P118 転写因子p53の特異的結合部位探索・認識機構 : マルチスケールシミュレーション研究(04.核酸結合蛋白質,ポスター,日本生物物理学会年会第51回(2013年度))

3P118 転写因子p53の特異的結合部位探索・認識機構 : マルチスケールシミュレーション研究(04.核酸結合蛋白質,ポスター,日本生物物理学会年会第51回(2013年度))

Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency

Type II restriction endonucleases (REases) are one of the basic tools of recombinant DNA technology. They also serve as models for elucidation of mechanisms for both site-specific DNA recognition and cleavage by proteins. However, isolation of catalytically active mutants from their libraries is challenging due to the toxicity of REases in the absence of protecting methylation, and techniques explored so far had limited success. Here, we present an improved SOS induction-based approach for in vivo screening of active REases, which we used to isolate a set of active variants of the catalytic mutant, Cfr10IE204Q. Detailed characterization of plasmids from 64 colonies screened from the library of ∼200 000 transformants revealed 29 variants of cfr10IR gene at the level of nucleotide sequence and 15 variants at the level of amino acid sequence, all of which were able to induce SOS response. Specific activity measurements of affinity-purified mutants revealed >200-fold variance among them, ranging from 100% (wild-type isolates) to 0.5% (S188C mutant), suggesting that the technique is equally suited for screening of mutants possessing high or low activity and confirming that it may be applied for identification of residues playing a role in catalysis.

DNA Interaction of the CcrM DNA Methyltransferase: A Mutational and Modeling Study

Caulobacter crescentus CcrM is a DNA-(adenine N6)-methyltransferase that methylates adenine in the sequence GANTC with high specificity. To investigate its mechanism of DNA recognition, we used the crystal structure of a related methyltransferase (M1.MboII, which modifies GAAGA) as a starting point, and docked into it a DNA substrate to identify the protein regions that approach the DNA. After alignment of CcrM and M1.MboII, we identified four candidate regions in CcrM to contain residues involved in DNA recognition. We mutated 20 amino acid residues within these regions, purified the CcrM variants, and determined their DNA-binding and catalytic activity on a cognate GANTC substrate and on nine near-cognate substrates, each of which contained a single base-pair substitution in the recognition sequence. Altogether, we identified four residues in two of the regions, mutations of which resulted in a strong (>100-fold) reduction of methylation activity. Our data show that DNA recognition by CcrM is a cooperative process, because disruption of critical contacts led to loss of catalytic activity but not to a relaxation in specificity. In addition, we identified a change in the readout of the fifth base pair in the GANTC sequence with two other CcrM variants that showed smaller reductions in overall activity. Based on this and the sequence alignment of CcrM with other DNA methyltransferases of same or related recognition sequence, we propose roles for these two regions in DNA recognition by CcrM.

Molecular mechanisms of Innate DNA recognition by the AIM2 and IFI16 inflammasomes (68.4)

Abstract Structures of The HIN Domain:DNA Complexes Reveal Ligand Binding and Activation Mechanisms of The AIM2 Inflammasome and IFI16

Purification, crystallization and preliminary X-ray analysis of OsAREB8 from rice, a member of the AREB/ABF family of bZIP transcription factors, in complex with its cognate DNA.

The AREB/ABF family of bZIP transcription factors play a key role in drought stress response and tolerance during the vegetative stage in plants. To reveal the DNA-recognition mechanism of the AREB/ABF family of proteins, the bZIP domain of OsAREB8, an AREB/ABF-family protein from Oryza sativa, was expressed in Escherichia coli, purified and crystallized with its cognate DNA. Crystals of the OsAREB8-DNA complex were obtained by the sitting-drop vapour-diffusion method at 277 K with a reservoir solution consisting of 50 mM MES pH 6.4, 29% MPD, 2 mM spermidine, 20 mM magnesium acetate and 100 mM sodium chloride. A crystal diffracted X-rays to 3.65 Å resolution and belonged to space group C222, with unit-cell parameters a = 155.1, b = 206.7, c = 38.5 Å. The crystal contained one OsAREB8-DNA complex in the asymmetric unit.

Structure-Based Models to Understand the Cipher that Governs Tale Protein Affinity with DNA.

Recent use of TALEs, transcription activator like effectors as a novel DNA binding domain, has raised the interest in deciphering the mechanisms of DNA recognition by these multimeric TALE proteins. TALE proteins can be fused with the catalytic domain of FOKI nucleases to act as artificial restriction enzymes to create a targeted DNA double strand break in-vivo for genome editing. The TAL effectors are characterized by a central domain of 30 tandem repeats of 33 to 35 amino acid sequence motifs. The amino acid sequence of each repeat is largely invariant, with the exception of two adjacent amino acid at position 12th and 13th (the repeat variable diresidue or RVD). Repeats with different RVD recognize different DNA base pairs, and it is hypothesized that there is one-to-one correspondence between the RVDs in the repeat domain and the nucleotide in the target DNA sequence. But how this central domain contacts DNA and what determines the specificity has remained enigmatic so far. Here we use structure-based models to investigate the structure of TALENS and their interaction with DNA. Simulations with biophysical models for multimeric protein in complex with DNA are presently too computationally expensive to help, but structure-based simulations been used to study protein folding and protein-protein interaction mechanisms for over a decade. We performed homology modeling to construct a structure of single repeat and 1.5 repeat using the PthA (TPR domain) as a template protein, which predicts only alpha helices and turns for both the repeat domains. Strucuture based models are generated by inspecting the native contacts of DNA and individual repeat domain from previous experimental knowledge and can be easily adapted to different DNA sequences and multidomain of TALE, as long as the binding motives are conserved.

Atypical DNA recognition mechanism used by the EspR virulence regulator of Mycobacterium tuberculosis

The human pathogen Mycobacterium tuberculosis requires the ESX-1 secretion system for full virulence. EspR plays a key role in ESX-1 regulation via direct binding and transcriptional activation of the espACD operon. Here, we describe the crystal structures of EspR, a C-terminally truncated form, EspRΔ10, as well as an EspR-DNA complex. EspR forms a dimer with each monomer containing an N-terminal helix-turn-helix DNA binding motif and an atypical C-terminal dimerization domain. Structural studies combined with footprinting experiments, atomic force microscopy and molecular dynamic simulations allow us to propose a model in which a dimer of EspR dimers is the minimal functional unit with two subunits binding two consecutive major grooves. The other two DNA binding domains are thus free to form higher-order oligomers and to bridge distant DNA sites in a cooperative way. These features are reminiscent of nucleoid-associated proteins and suggest a more general regulatory role for EspR than was previously suspected.

The particular mechanisms of DNA recognition and dimerization of MITF

The particular mechanisms of DNA recognition and dimerization of MITF