Validation of a clinical evaluation score for irritative dermatitis: SCOREPI.

BACKGROUND: Most dermatological conditions can be evaluated using validate clinical scores, no such tool is available for irritant contact dermatitis (ICD). OBJECTIVE: To create and validate a grid-based ICD severity score. METHODS: Three dermatologists developed the SCOre de REparation de l'EPIderme (SCOREPI) grid. Two studies were conducted to validate the SCOREPI. A cross-sectional study assessed the intra- and inter-observer error associated with using the SCOREPI. Each investigator received 15 min of training on proper use of the SCOREPI. A computer displayed a series of 20 photos of ICD, each of which were repeated three times in a randomized order. The prospective study assessed the correlation between SCOREPI with the severity of clinical symptoms as well as the sensitivity of the score to changes in ICD in response to topical treatment. RESULTS: The SCOREPI took an average of 35 ± 5 s to be completed and was characterized by an excellent intra-observer and moderate inter-observer reproducibility (intra-class correlation coefficient = 0.93 and 0.74, respectively). Significant divergence was observed between the physicians' assessment of estimated surface (P = 0.04), the presence of erythema (P < 0.0001) and the number of deep cracks (P = 0.0008). In the prospective analysis of patients, SCOREPI was correlated with tightness (r = 0.45; P < 0.0001), pain (r = 0.45; P < 0.0001), burning (r = 0.42; P < 0.0001), and pruritus (r = 0.28; P = 0.0055). SCOREPI decreased considerably during follow-up from 10.45 ± 4.61 to 4.82 ± 4.15 (P < 0.0001). CONCLUSION: The SCOREPI is easy to use, sensitive to change, and characterized by high intra- and moderate inter-observer reliability.

Mechanisms of chiral discrimination by topoisomerase IV.

Topoisomerase IV (Topo IV), an essential ATP-dependent bacterial type II topoisomerase, transports one segment of DNA through a transient double-strand break in a second segment of DNA. In vivo, Topo IV unlinks catenated chromosomes before cell division and relaxes positive supercoils generated during DNA replication. In vitro, Topo IV relaxes positive supercoils at least 20-fold faster than negative supercoils. The mechanisms underlying this chiral discrimination by Topo IV and other type II topoisomerases remain speculative. We used magnetic tweezers to measure the relaxation rates of single and multiple DNA crossings by Topo IV. These measurements allowed us to determine unambiguously the relative importance of DNA crossing geometry and enzymatic processivity in chiral discrimination by Topo IV. Our results indicate that Topo IV binds and passes DNA strands juxtaposed in a nearly perpendicular orientation and that relaxation of negative supercoiled DNA is perfectly distributive. Together, these results suggest that chiral discrimination arises primarily from dramatic differences in the processivity of relaxing positive and negative supercoiled DNA: Topo IV is highly processive on positively supercoiled DNA, whereas it is perfectly distributive on negatively supercoiled DNA. These results provide fresh insight into topoisomerase mechanisms and lead to a model that reconciles contradictory aspects of previous findings while providing a framework to interpret future results.

Tracking topoisomerase activity at the single-molecule level.

The recent development of new techniques to manipulate single DNA molecules has opened new opportunities for the study of the enzymes that control DNA topology: the type I and II topoisomerases. These single-molecule assays provide a unique way to study the uncoiling of single supercoiled DNA molecules and the unlinking of two intertwined DNAs. They allow for a detailed characterization of the activity of topoisomerases, including the processivity, the chiral discrimination, and the dependence of their enzymatic rate on ATP concentration, degree of supercoiling, and the tension in the molecule. These results shed new light on the mechanism of these enzymes and their function in vivo.

Topoisomerase IV bends and overtwists DNA upon binding.

Escherichia coli topoisomerase IV (Topo IV) is an essential ATP-dependent enzyme that unlinks sister chromosomes during replication and efficiently removes positive but not negative supercoils. In this article, we investigate the binding properties of Topo IV onto DNA in the absence of ATP using a single molecule micromanipulation setup. We find that the enzyme binds cooperatively (Hill coefficient alpha approximately 4) with supercoiled DNA, suggesting that the Topo IV subunits assemble upon binding onto DNA. It interacts preferentially with (+) rather than (-) supercoiled DNA (Kd+=0.15 nM, Kd-=0.23 nM) and more than two orders-of-magnitude more weakly with relaxed DNA (Kd0 approximately 36 nM). Like gyrase but unlike the eukaryotic Topo II, Topo IV bends DNA with a radius 0= 6.4 nm and locally changes its twist and/or its writhe by 0.16 turn per bound complex. We estimate its free energy of binding and study the dynamics of interaction of Topo IV with DNA at the binding threshold. We find that the protein/DNA complex alternates between two states: a weakly bound state where it stays with probability p = 0.89 and a strongly bound state (with probability p = 0.11). The methodology introduced here to characterize the Topo IV/DNA complex is very general and could be used to study other DNA/protein complexes.

Braiding DNA: experiments, simulations, and models.

DNA encounters topological problems in vivo because of its extended double-helical structure. As a consequence, the semiconservative mechanism of DNA replication leads to the formation of DNA braids or catenanes, which have to be removed for the completion of cell division. To get a better understanding of these structures, we have studied the elastic behavior of two braided nicked DNA molecules using a magnetic trap apparatus. The experimental data let us identify and characterize three regimes of braiding: a slightly twisted regime before the formation of the first crossing, followed by genuine braids which, at large braiding number, buckle to form plectonemes. Two different approaches support and quantify this characterization of the data. First, Monte Carlo (MC) simulations of braided DNAs yield a full description of the molecules' behavior and their buckling transition. Second, modeling the braids as a twisted swing provides a good approximation of the elastic response of the molecules as they are intertwined. Comparisons of the experiments and the MC simulations with this analytical model allow for a measurement of the diameter of the braids and its dependence upon entropic and electrostatic repulsive interactions. The MC simulations allow for an estimate of the effective torsional constant of the braids (at a stretching force F = 2 pN): C(b) approximately 48 nm (as compared with C approximately 100 nm for a single unnicked DNA). Finally, at low salt concentrations and for sufficiently large number of braids, the diameter of the braided molecules is observed to collapse to that of double-stranded DNA. We suggest that this collapse is due to the partial melting and fraying of the two nicked molecules and the subsequent right- or left-handed intertwining of the stretched single strands.

Statistical determination of the step size of molecular motors.

Molecular motors are enzymatic proteins that couple the consumption of chemical energy to mechanical displacement. In order to elucidate the translocation mechanisms of these enzymes, it is of fundamental importance to measure the physical step size. The step size can, in certain instances, be directly measured with single-molecule techniques; however, in the majority of cases individual steps are masked by noise. The step size can nevertheless be obtained from noisy single-molecule records through statistical methods. This analysis is analogous to determining the charge of the electron from current shot noise. We review methods for obtaining the step size based on analysing, in both the time and frequency domains, the variance in position from noisy single-molecule records of motor displacement. Additionally, we demonstrate how similar methods may be applied to measure the step size in bulk kinetic experiments.

Single-molecule study of DNA unlinking by eukaryotic and prokaryotic type-II topoisomerases.

Type-II topoisomerases are responsible for untangling DNA during replication by removing supercoiled and interlinked DNA structures. Using a single-molecule micromanipulation setup, we follow the real-time decatenation of two mechanically braided DNA molecules by Drosophila melanogaster topoisomerase (Topo) II and Escherichia coli Topo IV. Although Topo II relaxes left-handed (L) and right-handed (R-) braids similarly at a rate of approximately 2.9 s-1, Topo IV has a marked preference for L-braids, which it relaxes completely and processively at a rate of approximately 2.4 s-1. However, Topo IV can unlink R-braids at about half that rate when they supercoil to form L-plectonemes. These results imply that the preferred substrate for unlinking by Topo IV has the symmetry of an L-crossing and shed new light on the decatenation of daughter strands during DNA replication, which are usually assumed to be linked in an R-braid.

Thermophilic topoisomerase I on a single DNA molecule.

Control of DNA topology is critical in thermophilic organisms in which heightened ambient temperatures threaten the stability of the double helix. An important role in this control is played by topoisomerase I, a member of the type IA family of topoisomerases. We investigated the binding and activity of this topoisomerase from the hyperthermophilic bacterium Thermotoga maritima on duplex DNA using single molecule techniques, presenting it with various substrates such as (+) plectonemes, (-) plectonemes, and denaturation bubbles. We found the topoisomerase inactive on both types of plectonemes, but active on denaturation bubbles produced at increased stretching forces in underwound DNA. The relaxation rate depended sensitively on the applied force and the protein concentration. These observations could be understood in terms of a preference of the topoisomerase for single-stranded DNA over double-stranded DNA and allowed for a better understanding of activity of the topoisomerase in bulk experiments on circular plasmids. Binding experiments on a single duplex molecule using a mutant unable to perform cleavage confirmed this interpretation and suggested that T.maritima topoisomerase I behaves like an SSB by lowering the denaturation threshold of underwound DNA. Finally, experiments with a unique single-stranded DNA showed that both ends of the cleaved DNA are tightly maintained by the enzyme, supporting an enzyme-bridged mechanism for this topoisomerase.

Stretching single stranded DNA, a model polyelectrolyte.

The elastic properties of single stranded (ss)DNA, studied by pulling on an isolated molecule, are shown to agree with a recent model of ssDNA that takes into account base pairings and screened electrostatic repulsion of the phosphodiester backbone. By an appropriate physicochemical treatment, the pairing interactions were suppressed and ssDNA used as an experimental model for a generic polyelectrolyte. The elastic behavior of such an altered ssDNA deviates strongly from the behavior of an ideal polymer. This deviation is shown to result from the elasticity of the chain and its electrostatic self-avoiding interactions.

The mechanism of type IA topoisomerases.

The topology of cellular DNA is carefully controlled by enzymes called topoisomerases. By using single-molecule techniques, we monitored the activity of two type IA topoisomerases in real time under conditions in which single relaxation events were detected. The strict one-at-a-time removal of supercoils we observed establishes that these enzymes use an enzyme-bridged strand-passage mechanism that is well suited to their physiological roles and demonstrates a mechanistic unity with type II topoisomerases.

Twisting and stretching single DNA molecules.

The elastic properties of DNA are essential for its biological function. They control its bending and twisting as well as the induction of structural modifications in the molecule. These can affect its interaction with the cell machinery. The response of a single DNA molecule to a mechanical stress can be precisely determined in single-molecule experiments which give access to an accurate measurement of the elastic parameters of DNA.

Preferential relaxation of positively supercoiled DNA by E. coli topoisomerase IV in single-molecule and ensemble measurements.

We show that positively supercoiled [(+) SC] DNA is the preferred substrate for Escherichia coli topoisomerase IV (topo IV). We measured topo IV relaxation of (-) and (+) supercoils in real time on single, tethered DNA molecules to complement ensemble experiments. We find that the preference for (+) SC DNA is complete at low enzyme concentration. Otherwise, topo IV relaxed (+) supercoils at a 20-fold faster rate than (-) supercoils, due primarily to about a 10-fold increase in processivity with (+) SC DNA. The preferential cleavage of (+) SC DNA in a competition experiment showed that substrate discrimination can take place prior to strand passage in the presence or absence of ATP. We propose that topo IV discriminates between (-) and (+) supercoiled DNA by recognition of the geometry of (+) SC DNA. Our results explain how topo IV can rapidly remove (+) supercoils to support DNA replication without relaxing the essential (-) supercoils of the chromosome. They also show that the rate of supercoil relaxation by topo IV is several orders of magnitude faster than hitherto appreciated, so that a single enzyme may suffice at each replication fork.

Replication by a single DNA polymerase of a stretched single-stranded DNA.

A new approach to the study of DNA/protein interactions has been opened through the recent advances in the manipulation of single DNA molecules. These allow the behavior of individual molecular motors to be studied under load and compared with bulk measurements. One example of such a motor is the DNA polymerase, which replicates DNA. We measured the replication rate by a single enzyme of a stretched single strand of DNA. The marked difference between the elasticity of single- and double-stranded DNA allows for the monitoring of replication in real time. We have found that the rate of replication depends strongly on the stretching force applied to the template. In particular, by varying the load we determined that the biochemical steps limiting replication are coupled to movement. The replication rate increases at low forces, decreases at forces greater than 4 pN, and ceases when the single-stranded DNA substrate is under a load greater than approximately 20 pN. The decay of the replication rate follows an Arrhenius law and indicates that multiple bases on the template strand are involved in the rate-limiting step of each cycle. This observation is consistent with the induced-fit mechanism for error detection during replication.

Stress-induced structural transitions in DNA and proteins.

The ability to manipulate, stretch and twist biomolecules opens the way to an understanding of their structural transitions. We review some of the recently discovered stress-induced structural transitions in DNA as well as the application of single molecule manipulation techniques to DNA unzipping and to the study of protein folding/unfolding transitions.

Single-molecule analysis of DNA uncoiling by a type II topoisomerase.

Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment. This enables them to untangle DNA and relax the interwound supercoils (plectonemes) that arise in twisted DNA. In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death. Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled. By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the damping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.

[Human globin genes: what can we learn from their polymorphism?]. / Les gènes des globines humaines: que nous apprend leur polymorphisme?

Molecular analysis of human alpha and beta globin genes reveals extensive polymorphism at these loci. Worldwide distribution of the sickle cell trait has been well known for some time. However, the molecular basis and distribution of thalassemia have been more recently studied. These are the commonest monogenic disorders. For most of them, beta-thalassemia is due to single nucleotide substitutions, small deletions or insertions. They are very heterogeneous and widely dispersed in the Old World. alpha-thalassemia is mainly due to the deletion of one to four alpha genes. On the whole, their distribution is quite similar to beta-thalassemia. With some exceptions, both distributions coincide with present and past regions of malarious endemicity. On the other hand, when looking at individual mutations, no two regions are identical. The question of whether selection by malaria plays a role on observed allele frequencies is still a challenge. The only well clear instance is the beta S mutations, which causes sickle cell anaemia. The role of malaria is but one among other hypothesis for explaining thalassemia distribution and frequencies. A possible scenario could be the following: one (or a few) mutation happened in a population and spread because of its selective advantage, along with the founder effect and/or genetic drift. Migration, founder effect and genetic drift must be invoked to account for some observations. It is still difficult to say why a mutation is highly frequent in one population and not in another., even at equivalent malarial endemicity. On the other hand, many genes should contribute simultaneously, or in synergy, in the process of fighting against malaria. Fitness of each mutation could depend on its genetic background when the mutation arose. Selection must work on a set of genes. Populations who are living now, and genetically very different, could very well be the result of selection on many genes by many infectious agents.

Micro-mechanical measurement of the torsional modulus of DNA.

The torsional modulus C of DNA is determined from the difference between the work of stretching a single overwound molecule and the work done in stretching one underwound by the same number of turns. The value obtained C/kBT = 86 +/- 10 nm is within the range (75 +/- 25 nm) estimated by more indirect methods.

Fast combinatorial cartography by FISH on combed genomic DNA.

A novel combinatorial method combining FISH on combed genomic DNA is presented for a fast high-resolution (1 Kbps) gene cartography.

Stretched and overwound DNA forms a Pauling-like structure with exposed bases.

We investigate structural transitions within a single stretched and supercoiled DNA molecule. With negative supercoiling, for a stretching force >0.3 pN, we observe the coexistence of B-DNA and denatured DNA from sigma approximately -0.015 down to sigma = -1. Surprisingly, for positively supercoiled DNA (sigma > +0.037) stretched by 3 pN, we observe a similar coexistence of B-DNA and a new, highly twisted structure. Experimental data and molecular modeling suggest that this structure has approximately 2.62 bases per turn and an extension 75% larger than B-DNA. This structure has tightly interwound phosphate backbones and exposed bases in common with Pauling's early DNA structure [Pauling, L. & Corey, R. B. (1953), Proc. Natl. Acad. Sci. USA 39, 84-97] and an unusual structure proposed for the Pf1 bacteriophage [Liu, D. J. & Day, L. A. (1994) Science 265, 671-674].