Novel insights on the DNA interaction of calicheamicin γ1(I).

Calicheamicin γ1(I) (Cal) is a unique molecule in which a DNA binding motif (aryl-tetrasaccharide) is linked to a DNA cleaving moiety (calicheamicinone). The hallmark of this natural product rests in the impressive optimization of these two mechanisms leading to a drug that is extremely efficient in cleaving DNA at well-defined sites. However, the relative contributions of these two structurally distinct domains to the overall process have not been fully elucidated yet. Here, we used different experimental approaches to better dissect the role of the aryl-tetrasaccharide and the enediyne moieties in the DNA sequence selective binding step as well as the in the cleavage reaction. Our results highlight the remarkable cooperation of the two components in producing an amazing molecular machine. The herein presented molecular details of this concerted mechanism of action can be further applied to rationally design more druggable compounds.

Long-range mapping of DNA.

Sequence-specific optical signals are used to establish long-range sequence order and identification for fragments hundreds of kilo bases in length.

Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA.

We have investigated the anomalously weak binding of human papillomavirus (HPV) regulatory protein E2 to a DNA target containing the spacer sequence TATA. Experiments in magnesium (Mg(2+)) and calcium (Ca(2+)) ion buffers revealed a marked reduction in cutting by DNase I at the CpG sequence in the protein-binding site 3' to the TATA spacer sequence, Studies of the cation dependence of DNA-E2 affinities showed that upon E2 binding the TATA sequence releases approximately twice as many Mg(2+) ions as the average of the other spacer sequences. Binding experiments for TATA spacer relative to ATAT showed that in potassium ion (K(+)) the E2 affinity of the two sequences is nearly equal, but the relative dissociation constant (K(d)) for TATA increases in the order K(+ )< Na(+ )< Ca(2+ )< Mg(2+). Except for Mg(2+), K(d) for TATA relative to ATAT is independent of ion concentration, whereas for Mg(2+) the affinity for TATA drops sharply as ion concentration increases. Thus, ions of increasing positive charge density increasingly distort the E2 binding site, weakening the affinity for protein. In the case of Mg(2+), additional ions are bound to TATA that require displacement for protein binding. We suggest that the TATA sequence may bias the DNA structure towards a conformation that binds the protein relatively weakly.

The role of nucleotide cofactor binding in cooperativity and specificity of MutS recognition.

Mismatch repair (MMR) is essential for eliminating biosynthetic errors generated during replication or genetic recombination in virtually all organisms. The critical first step in Escherichia coli MMR is the specific recognition and binding of MutS to a heteroduplex, containing either a mismatch or an insertion/deletion loop of up to four nucleotides. All known MutS homologs recognize a similar broad spectrum of substrates. Binding and hydrolysis of nucleotide cofactors by the MutS-heteroduplex complex are required for downstream MMR activity, although the exact role of the nucleotide cofactors is less clear. Here, we showed that MutS bound to a 30-bp heteroduplex containing an unpaired T with a binding affinity approximately 400-fold stronger than to a 30-bp homoduplex, a much higher specificity than previously reported. The binding of nucleotide cofactors decreased both MutS specific and nonspecific binding affinity, with the latter marked by a larger drop, further increasing MutS specificity by approximately 3-fold. Kinetic studies showed that the difference in MutS K(d) for various heteroduplexes was attributable to the difference in intrinsic dissociation rate of a particular MutS-heteroduplex complex. Furthermore, the kinetic association event of MutS binding to heteroduplexes was marked by positive cooperativity. Our studies showed that the positive cooperativity in MutS binding was modulated by the binding of nucleotide cofactors. The binding of nucleotide cofactors transformed E. coli MutS tetramers, the functional unit in E. coli MMR, from a cooperative to a noncooperative binding form. Finally, we found that E. coli MutS bound to single-strand DNA with significant affinity, which could have important implication for strand discrimination in eukaryotic MMR mechanism.

Stepwise binding and bending of DNA by Escherichia coli integration host factor.

Integration host factor (IHF) is a prokaryotic protein required for the integration of lambda phage DNA into its host genome. An x-ray crystal structure of the complex shows that IHF binds to the minor groove of DNA and bends the double helix by 160 degrees [Rice PA, Yang S, Mizuuchi K, Nash HA (1996) Cell 87:1295-1306]. We sought to dissect the complex formation process into its component binding and bending reaction steps, using stopped-flow fluorimetry to observe changes in resonance energy transfer between DNA-bound dyes, which in turn reflect distance changes upon bending. Different DNA substrates that are likely to increase or decrease the DNA bending rate were studied, including one with a nick in a critical kink position, and a substrate with longer DNA ends to increase hydrodynamic friction during bending. Kinetic experiments were carried out under pseudofirst-order conditions, in which the protein concentration is in substantial excess over DNA. At lower concentrations, the reaction rate rises linearly with protein concentration, implying rate limitation by the bimolecular reaction step. At high concentrations the rate reaches a plateau value, which strongly depends on temperature and the nature of the DNA substrate. We ascribe this reaction limit to the DNA bending rate and propose that complex formation is sequential at high concentration: IHF binds rapidly to DNA, followed by slower DNA bending. Our observations on the bending step kinetics are in agreement with results using the temperature-jump kinetic method.

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF.

Regulation of gene expression involves formation of specific protein-DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNA-binding sites. Previous kinetics measurements on several DNA-bending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending/unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from approximately 2 ms(-1) at approximately 37 degrees C to approximately 40 ms(-1) at approximately 10 degrees C and correspond to an activation energy of approximately 14 +/- 3 kcal/mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend/kink DNA before forming a tight complex with IHF.

Statistical-mechanical theory of DNA looping.

The lack of a rigorous analytical theory for DNA looping has caused many DNA-loop-mediated phenomena to be interpreted using theories describing the related process of DNA cyclization. However, distinctions in the mechanics of DNA looping versus cyclization can have profound quantitative effects on the thermodynamics of loop closure. We have extended a statistical mechanical theory recently developed for DNA cyclization to model DNA looping, taking into account protein flexibility. Notwithstanding the underlying theoretical similarity, we find that the topological constraint of loop closure leads to the coexistence of multiple classes of loops mediated by the same protein structure. These loop topologies are characterized by dramatic differences in twist and writhe; because of the strong coupling of twist and writhe within a loop, DNA looping can exhibit a complex overall helical dependence in terms of amplitude, phase, and deviations from uniform helical periodicity. Moreover, the DNA-length dependence of optimal looping efficiency depends on protein elasticity, protein geometry, and the presence of intrinsic DNA bends. We derive a rigorous theory of loop formation that connects global mechanical and geometric properties of both DNA and protein and demonstrates the importance of protein flexibility in loop-mediated protein-DNA interactions.

Analysis of in-vivo LacR-mediated gene repression based on the mechanics of DNA looping.

Interactions of E. coli lac repressor (LacR) with a pair of operator sites on the same DNA molecule can lead to the formation of looped nucleoprotein complexes both in vitro and in vivo. As a major paradigm for loop-mediated gene regulation, parameters such as operator affinity and spacing, repressor concentration, and DNA bending induced by specific or non-specific DNA-binding proteins (e.g., HU), have been examined extensively. However, a complete and rigorous model that integrates all of these aspects in a systematic and quantitative treatment of experimental data has not been available. Applying our recent statistical-mechanical theory for DNA looping, we calculated repression as a function of operator spacing (58-156 bp) from first principles and obtained excellent agreement with independent sets of in-vivo data. The results suggest that a linear extended, as opposed to a closed v-shaped, LacR conformation is the dominant form of the tetramer in vivo. Moreover, loop-mediated repression in wild-type E. coli strains is facilitated by decreased DNA rigidity and high levels of flexibility in the LacR tetramer. In contrast, repression data for strains lacking HU gave a near-normal value of the DNA persistence length. These findings underscore the importance of both protein conformation and elasticity in the formation of small DNA loops widely observed in vivo, and demonstrate the utility of quantitatively analyzing gene regulation based on the mechanics of nucleoprotein complexes.

The kinetics of ligand binding by an adenine-sensing riboswitch.

A riboswitch within the 5' untranslated region (UTR) of the Bacillus subtilis pbuE mRNA binds adenine and related analogues in the absence of protein factors; excess adenine added to bacterial growth media triggers activation of a reporter gene that carries this riboswitch. To assess whether the riboswitch reaches thermodynamic equilibrium, or is operated by the kinetics of ligand binding and RNA transcription, we examined the detailed equilibrium and kinetic parameters for the complex formation between the aptamer domain of this riboswitch and the ligands adenine, 2-aminopurine (2AP), and 2,6-diaminopurine (DAP). Using a fluorescence-based assay, we have confirmed that adenine and 2AP have nearly equal binding affinity, with KD values for 2AP ranging from 250 nM to 3 microM at temperatures ranging from 15 to 35 degrees C, while DAP binds with much higher affinity. The association rate constant, however, favors adenine over DAP and 2AP by 3- and 10-fold, respectively, at 25 degrees C. Furthermore, the rate constants for adenine association and dissociation with the aptamer suggest that the pbuE riboswitch could be either kinetically or thermodynamically controlled depending upon the time scale of transcription and external variables such as temperature. We cite data that suggest kinetic control under certain conditions and illustrate with a model calculation how the system can switch between kinetic and equilibrium control. These findings further support the hypothesis that many riboswitches rely on the kinetics of ligand binding and the speed of RNA transcription, rather than simple ligand affinity, to establish the concentration of metabolite needed to trigger riboswitch function.

The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch.

Riboswitches are genetic control elements that usually reside in untranslated regions of messenger RNAs. These folded RNAs directly bind metabolites and undergo allosteric changes that modulate gene expression. A flavin mononucleotide (FMN)-dependent riboswitch from the ribDEAHT operon of Bacillus subtilis uses a transcription termination mechanism wherein formation of an RNA-FMN complex causes formation of an intrinsic terminator stem. We assessed the importance of RNA transcription speed and the kinetics of FMN binding to the nascent mRNA for riboswitch function. The riboswitch does not attain thermodynamic equilibrium with FMN before RNA polymerase needs to make a choice between continued transcription and transcription termination. Therefore, this riboswitch is kinetically driven, and functions more like a "molecular fuse." This reliance on the kinetics of ligand association and RNA polymerization speed might be common for riboswitches that utilize transcription termination mechanisms.

Predicting indirect readout effects in protein-DNA interactions.

Recognition of DNA by proteins relies on direct interactions with specific DNA-functional groups, along with indirect effects that reflect variable energetics in the response of DNA sequences to twisting and bending distortions induced by proteins. Predicting indirect readout requires knowledge of the variations in DNA curvature and flexibility in the affected region, which we have determined for a series of DNA-binding sites for the E2 regulatory protein by using the cyclization kinetics method. We examined 16 sites containing different noncontacted spacer sequences, which vary by more than three orders of magnitude in binding affinity. For 15 of these sites, the variation in affinity was predicted within a factor of 3, by using experimental curvature and flexibility values and a statistical mechanical theory. The sole exception was traced to differential magnesium ion binding.

High-throughput approach for detection of DNA bending and flexibility based on cyclization.

We have developed a high-throughput approach to the labor-intensive problems of DNA cyclization, which we use to characterize DNA curvature and mechanical properties. The method includes a combinatorial approach to make the DNA constructs needed and automated real-time measurement of the kinetics using fluorescence. We validated the approach and investigated the flexibility of two kinds of nicked DNA and AT dinucleotide repeats. We found that, although the nicks hardly alter the bending flexibility, they significantly increase the torsional flexibility, and that the AT repeat has 28% (+/-12%) lower bending rigidity than a generic DNA sequence.

Structural origins of adenine-tract bending.

DNA sequences containing short adenine tracts are intrinsically curved and play a role in transcriptional regulation. Despite many high-resolution NMR and x-ray studies, the origins of curvature remain disputed. Long-range restraints provided by 85 residual dipolar couplings were measured for a DNA decamer containing an adenine (A)(4)-tract and used to refine the structure. The overall bend in the molecule is a result of in-phase negative roll in the A-tract and positive roll at its 5' junction, as well as positive and negative tilt inside the A-tract and near its junctions. The bend magnitude and direction obtained from NMR structures is 9.0 degrees into the minor groove in a coordinate frame located at the third AT base pair. We evaluated long-range and wedge models for DNA curvature and concluded that our data for A-tract curvature are best explained by a "delocalized bend" model. The global bend magnitude and direction of the NMR structure are in excellent agreement with the junction model parameters used to rationalize gel electrophoretic data and with preliminary results of a cyclization kinetics assay from our laboratory.

Statistical mechanics of sequence-dependent circular DNA and its application for DNA cyclization.

DNA cyclization is potentially the most powerful approach for systematic quantitation of sequence-dependent DNA bending and flexibility. We extend the statistical mechanics of the homogeneous DNA circle to a model that considers discrete basepairs, thus allowing for inhomogeneity, and apply the model to analysis of DNA cyclization. The theory starts from an iterative search for the minimum energy configuration of circular DNA. Thermodynamic quantities such as the J factor, which is essentially the ratio of the partition functions of circular and linear forms, are evaluated by integrating the thermal fluctuations around the configuration under harmonic approximation. Accurate analytic expressions are obtained for equilibrium configurations of homogeneous circular DNA with and without bending anisotropy. J factors for both homogeneous and inhomogeneous DNA are evaluated. Effects of curvature, helical repeat, and bending and torsional flexibility in DNA cyclization are analyzed in detail, revealing that DNA cyclization can detect as little as one degree of curvature and a few percent change in flexibility. J factors calculated by our new approach are well consistent with Monte Carlo simulations, whereas the new theory has much greater efficiency in computations. Simulation of experimental results has been demonstrated.

Bending and flexibility of methylated and unmethylated EcoRI DNA.

We used cyclization kinetics experiments and Monte Carlo simulations to determine a structural model for a DNA decamer containing the EcoRI restriction site. Our findings agree well with recent crystal and NMR structures of the EcoRI dodecamer, where an overall bend of seven degrees is distributed symmetrically over the molecule. Monte Carlo simulations indicate that the sequence has a higher flexibility, assumed to be isotropic, compared to that of a "generic" DNA sequence. This model was used as a starting point for the investigation of the effect of cytosine methylation on DNA bending and flexibility. While methylation did not affect bend magnitude or direction, it resulted in a reduction in bending flexibility and under-winding of the methylated nucleotides. We demonstrate that our approach can augment the understanding of DNA structure and dynamics by adding information about the global structure and flexibility of the sequence. We also show that cyclization kinetics can be used to study the properties of modified nucleotides.

Concerted binding and bending of DNA by Escherichia coli integration host factor.

Integration host factor (IHF) is a heterodimeric Escherichia coli protein that plays essential roles in a variety of cellular processes including site-specific recombination, transcription, and DNA replication. The IHF-DNA interface extends over three helical turns and includes sequential minor groove contacts that present strong, sequence specific protection patterns against hydroxyl radical cleavage. Synchrotron X-ray footprinting has been used to follow the kinetics of formation of DNA-protein contacts in the IHF-DNA complex with single base-pair spatial, and millisecond time, resolution. The three sites of IHF protection on the DNA develop with similar time-dependence, indicating that sequence specific binding and bending occur concertedly. Two distinct phases are observed in the association process. The first "burst" phase is characterized by a rate that is greater than diffusion limited (>10(10) s(-1) M(-1)) and the second phase is on the order of diffusion controlled (approximately 10(8) M(-1) s(-1)). The overall kinetics of association become faster with increasing IHF concentration showing that complex formation is second-order with protein. The rate of association is maximal between 100 and 200 mM KCl decreasing at higher and lower concentrations. The rate of IHF dissociation from site-specifically bound DNA increases monotonically as KCl concentration is increased. The dissociation progress curves are biphasic with the amplitude of the first phase dependent upon competitor DNA concentration. These results are the first analysis by synchrotron footprinting of the fast kinetics of a protein-DNA interaction and suggest that IHF binds its specific site through a multiple-step mechanism in which the first step is facilitated diffusion along the length of the duplex followed by subsequent binding and bending of the DNA in a concerted manner.

Calculation of binding isotherms for heterogeneous polymers.

The matrix method of statistical mechanics is used to calculate equilibria for the binding of small molecules to polymers. When there is only one kind of binding site the problem is simple; some examples are given for illustrative purposes. If, however, the binding sites are not all equivalent and the bound molecules interact or interfere with each other, the problem is no longer trivial, being formally analogous with calculation of the helix-coil transition equilibrium in a heterogeneous polypeptide. Particular difficulties arise when the sequence of binding sites is aperiodic; most naturally occurring materials fall in this class. The purpose of this paper is to point out that problems of this type are readily solved with good accuracy by use of random-number methods on a high-speed digital computer. One such calculation is presented for illustration. The methods developed are applicable to such systems as the binding of actinomycin, Hg- , and acridine dyes to DNA.