A programmable DNA roadblock system using dCas9 and multivalent target sites.

A protein roadblock forms when a protein binds DNA and hinders translocation of other DNA binding proteins. These roadblocks can have significant effects on gene expression and regulation as well as DNA binding. Experimental methods for studying the effects of such roadblocks often target endogenous sites or introduce non-variable specific sites into DNAs to create binding sites for artificially introduced protein roadblocks. In this work, we describe a method to create programmable roadblocks using dCas9, a cleavage deficient mutant of the CRISPR effector nuclease Cas9. The programmability allows us to custom design target sites in a synthetic gene intended for in vitro studies. These target sites can be coded with multivalency-in our case, internal restriction sites which can be used in validation studies to verify complete binding of the roadblock. We provide full protocols and sequences and demonstrate how to use the internal restriction sites to verify complete binding of the roadblock. We also provide example results of the effect of DNA roadblocks on the translocation of the restriction endonuclease NdeI, which searches for its cognate site using one dimensional diffusion along DNA.

Salt concentration modulates the DNA target search strategy of NdeI.

DNA target search is a key step in cellular transactions that access genomic information. How DNA binding proteins combine 3D diffusion, sliding and hopping into an overall search strategy remains poorly understood. Here we report the use of a single molecule DNA tethering method to characterize the target search kinetics of the type II restriction endonuclease NdeI. The measured search rate depends strongly on DNA length as well as salt concentration. Using roadblocks, we show that there are significant changes in the DNA sliding length over the salt concentrations in our study. To explain our results, we propose a model including cycles of 3D and 1D search in which salt concentration modulates the strategy by varying the length of DNA probed per 1D scan. At low salt NdeI makes a single non-specific encounter with DNA followed by an effective and complete 1D scan. At higher salt, NdeI must execute multiple cycles of target search due to the reduced efficacy of 1D search.

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage.

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

The Role of Noncognate Sites in the 1D Search Mechanism of EcoRI.

A one-dimensional (1D) search is an essential step in DNA target recognition. Theoretical studies have suggested that the sequence dependence of 1D diffusion can help resolve the competing demands of a fast search and high target affinity, a conflict known as the speed-selectivity paradox. The resolution requires that the diffusion energy landscape is correlated with the underlying specific binding energies. In this work, we report observations of a 1D search by quantum dot-labeled EcoRI. Our data supports the view that proteins search DNA via rotation-coupled sliding over a corrugated energy landscape. We observed that whereas EcoRI primarily slides along DNA at low salt concentrations, at higher concentrations, its diffusion is a combination of sliding and hopping. We also observed long-lived pauses at genomic star sites, which differ by a single nucleotide from the target sequence. To reconcile these observations with prior biochemical and structural data, we propose a model of search in which the protein slides over a sequence-independent energy landscape during fast search but rapidly interconverts with a "hemispecific" binding mode in which a half site is probed. This half site interaction stabilizes the transition to a fully specific mode of binding, which can then lead to target recognition.

Analyzing dwell times with the Generalized Method of Moments.

The Generalized Method of Moments (GMM) is a statistical method for the analysis of samples from random processes. First developed for the analysis of econometric data, the method is here formulated to extract hidden kinetic parameters from measurements of single molecule dwell times. Our method is based on the analysis of cumulants of the measured dwell times. We develop a general form of an objective function whose minimization can return estimates of decay parameters for any number of intermediates directly from the data. We test the performance of our technique using both simulated and experimental data. We also compare the performance of our method to nonlinear least-squares minimization (NL-LSQM), a commonly-used technique for analysis of single molecule dwell times. Our findings indicate that the GMM performs comparably to NL-LSQM over most of the parameter range we explore. It offers some benefits compared with NL-LSQM in that it does not require binning, exhibits slightly lower bias and variance with small sample sizes (N<20), and is somewhat superior in identifying fast decay times with these same low count data sets. Additionally, a comparison with the Classical Method of Moments (CMM) shows that the CMM can fail in many cases, whereas the GMM always returns estimates. Our results show that the GMM can be a useful tool and complements standard approaches to analysis of single molecule dwell times.

A single molecule assay for measuring site-specific DNA cleavage.

Sequence-specific DNA cleavage is a key step in a number of genomic transactions. Here, we report a single-molecule technique that allows the simultaneous measurement of hundreds of DNAs, thereby collecting significant statistics in a single experiment. Microbeads are tethered with single DNA molecules in a microfluidic channel. After the DNA cleavage reaction is initiated, the time of cleavage of each DNA is recorded using video microscopy. We demonstrate the utility of our method by measuring the cleavage kinetics of NdeI, a type II restriction endonuclease.

DNA motion capture reveals the mechanical properties of DNA at the mesoscale.

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-µm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length.

Synthesis and characterization of a BODIPY-labeled derivative of Soraphen A that binds to acetyl-CoA carboxylase.

BODIPY-labeled Soraphen A derivative 4 was synthesized and characterized as an acetyl-CoA carboxylase (ACC) binder. Biophysical measurements indicate that the molecule binds in the biotin carboxylase domain where Soraphen A has been shown to bind. The fluorescent label of the BODIPY can be used to biophysically identify a compound that binds to the Soraphen A site of the biotin carboxylase domain versus the carboxytransferase domain of ACC.

The structure of the BIR3 domain of cIAP1 in complex with the N-terminal peptides of SMAC and caspase-9.

The inhibitor of apoptosis protein (IAP) family of molecules inhibit apoptosis through the suppression of caspase activity. It is known that the XIAP protein regulates both caspase-3 and caspase-9 through direct protein-protein interactions. Specifically, the BIR3 domain of XIAP binds to caspase-9 via a ;hotspot' interaction in which the N-terminal residues of caspase-9 bind in a shallow groove on the surface of XIAP. This interaction is regulated via SMAC, the N-terminus of which binds in the same groove, thus displacing caspase-9. The mechanism of suppression of apoptosis by cIAP1 is less clear. The structure of the BIR3 domain of cIAP1 (cIAP1-BIR3) in complex with N-terminal peptides from both SMAC and caspase-9 has been determined. The binding constants of these peptides to cIAP1-BIR3 have also been determined using the surface plasmon resonance technique. The structures show that the peptides interact with cIAP1 in the same way that they interact with XIAP: both peptides bind in a similar shallow groove in the BIR3 surface, anchored at the N-terminus by a charge-stabilized hydrogen bond. The binding data show that the SMAC and caspase-9 peptides bind with comparable affinities (85 and 48 nM, respectively).

Mutation of a conserved active site residue converts tyrosyl-DNA phosphodiesterase I into a DNA topoisomerase I-dependent poison.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3' and 5' phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1-DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.0 A crystal structure of yeast Tdp1 was determined that is very similar to human Tdp1. Poorly conserved regions of primary structure are peripheral to an essentially identical catalytic core. Enzyme mechanism was also conserved, because the yeast SCAN1 mutant (H(432)R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1-dependent lethality of Tdp1H(432)N was drug-independent, coinciding with increased covalent Top1-DNA and Tdp1-DNA complex formation in vivo. However, both H(432) mutants were recessive to wild-type Tdp1. Thus, yeast H(432) acts in the general acid/base catalytic mechanism of Tdp1 to resolve 3' phosphotyrosyl and 3' phosphoamide linkages. However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H(432)N-catalyzed resolution of 3' phospho-adducts.

Structure-activity relationships at the 5-position of thiolactomycin: an intact (5R)-isoprene unit is required for activity against the condensing enzymes from Mycobacterium tuberculosis and Escherichia coli.

Thiolactomycin inhibits bacterial cell growth through inhibition of the beta-ketoacyl-ACP synthase activity of type II fatty acid synthases. The effect of modifications of the 5-position isoprenoid side chain on both IC(50) and MIC were determined. Synthesis and screening of a structurally diverse set of 5-position analogues revealed very little tolerance for substitution in purified enzyme assays, but a few analogues retained MIC, presumably through another target. Even subtle modifications such as reducing one or both double bonds of the diene were not tolerated. The only permissible structural modifications were removal of the isoprene methyl group or addition of a methyl group to the terminus. Cocrystallization of these two inhibitors with the condensing enzyme from Escherichia coli revealed that they retained the TLM binding mode at the active site with reduced affinity. These results suggest a strict requirement for a conjugated, planar side chain inserting within the condensing enzyme active site.

Cofactor-induced conformational rearrangements establish a catalytically competent active site and a proton relay conduit in FabG.

beta-Ketoacyl-acyl carrier protein reductase (FabG) is a key component in the type II fatty acid synthase system. The structures of Escherichia coli FabG and the FabG[Y151F] mutant in binary complexes with NADP(H) reveal that mechanistically important conformational changes accompany cofactor binding. The active site Ser-Tyr-Lys triad is repositioned into a catalytically competent constellation, and a hydrogen bonded network consisting of ribose hydroxyls, the Ser-Tyr-Lys triad, and four water molecules creates a proton wire to replenish the tyrosine proton donated during catalysis. Also, a disordered loop in FabG forms a substructure in the complex that shapes the entrance to the active site. A key observation is that the nicotinamide portion of the cofactor is disordered in the FabG[Y151F].NADP(H) complex, and Tyr151 appears to be necessary for high-affinity cofactor binding. Biochemical data confirm that FabG[Y151F] is defective in NADPH binding. Finally, structural changes consistent with the observed negative cooperativity of FabG are described.

The 1.3-Angstrom-resolution crystal structure of beta-ketoacyl-acyl carrier protein synthase II from Streptococcus pneumoniae.

The beta-ketoacyl-acyl carrier protein synthases are members of the thiolase superfamily and are key regulators of bacterial fatty acid synthesis. As essential components of the bacterial lipid metabolic pathway, they are an attractive target for antibacterial drug discovery. We have determined the 1.3 A resolution crystal structure of the beta-ketoacyl-acyl carrier protein synthase II (FabF) from the pathogenic organism Streptococcus pneumoniae. The protein adopts a duplicated betaalphabetaalphabetaalphabetabeta fold, which is characteristic of the thiolase superfamily. The two-fold pseudosymmetry is broken by the presence of distinct insertions in the two halves of the protein. These insertions have evolved to bind the specific substrates of this particular member of the thiolase superfamily. Docking of the pantetheine moiety of the substrate identifies the loop regions involved in substrate binding and indicates roles for specific, conserved residues in the substrate binding tunnel. The active site triad of this superfamily is present in spFabF as His 303, His 337, and Cys 164. Near the active site is an ion pair, Glu 346 and Lys 332, that is conserved in the condensing enzymes but is unusual in our structure in being stabilized by an Mg(2+) ion which interacts with Glu 346. The active site histidines interact asymmetrically with Lys 332, whose positive charge is closer to His 303, and we propose a specific role for the lysine in polarizing the imidazole ring of this histidine. This asymmetry suggests that the two histidines have unequal roles in catalysis and provides new insights into the catalytic mechanisms of these enzymes.

A Phase I study of gemcitabine with concurrent radiotherapy in stage III, locally advanced non-small cell lung cancer.

PURPOSE: Our goal was to find the maximum tolerated dose of gemcitabine administered concurrently with thoracic radiotherapy in locally advanced non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with stage III NSCLC and a radiation planning volume less than 2000 cm(3) were included. Treatment consisted of 6 weeks of thoracic radiation, 2 Gy daily for 5 days a week for a total dose of 60 Gy. Planning with multiple field arrangements and three-dimensional conformal technique was used. Patients were treated with gemcitabine, starting with a dose of 300 mg/m(2) in the 1st week of radiation. In subsequent cohorts, the weekly dosing frequency of gemcitabine was increased until weekly administration was reached. Thereafter, the doses of weekly gemcitabine were increased. Toxicity was measured using Common Toxicity of the National Cancer Institute (CTC), acute Radiation Therapy Oncology Group (RTOG), and late RTOG/European Organization for Research and Treatment of Cancer (EORTC) rating scales. RESULTS: Twenty-seven patients were included, of whom 14 had stage IIIa and 13 had stage IIIb. Dose-limiting toxicity was grade 3 esophagitis and grade 3 radiation pneumonitis in the patient cohort receiving gemcitabine 450 mg/m(2) once weekly. The mean actual treated radiation volume was 760 cm(3) (range, 289-1718 cm(3)). CONCLUSIONS: The maximum tolerated dose and frequency of gemcitabine in locally advanced NSCLC is 300 mg/m(2) once weekly during 6 weeks of thoracic radiotherapy, as long as the treatment volume does not exceed 2000 cm(3).

A missense mutation in the fabB (beta-ketoacyl-acyl carrier protein synthase I) gene confers tiolactomycin resistance to Escherichia coli.

Thiolactomycin (TLM) is an antibiotic that inhibits bacterial type II fatty acid synthesis at the condensing enzyme step, and beta-ketoacyl-acyl carrier protein synthase I (FabB) is the relevant target in Escherichia coli. TLM resistance is associated with the upregulation of efflux pumps. Therefore, a tolC knockout mutant (strain ANS1) was constructed to eliminate the contribution of type I secretion systems to TLM resistance. Six independent TLM-resistant clones of strain ANS1 were isolated, and all possessed the same missense mutation in the fabB gene (T1168G) that directed the expression of a mutant protein, FabB(F390V). FabB(F390V) was resistant to TLM in vitro. Leucine is the only other amino acid found at position 390 in nature, and the Staphylococcus aureus FabF protein, which contains this substitution, was sensitive to TLM. Structural modeling predicted that the CG2 methyl group of the valine side chain interfered with the positioning of the C11 methyl on the isoprenoid side chain of TLM in the binary complex, whereas the absence of a bulky methyl group on the leucine side chain permitted TLM binding. These data illustrate that missense mutations that introduce valine at position 390 confer TLM resistance while maintaining the vital catalytic properties of FabB.