DNA-Topology Simplification by Topoisomerases.

The topological properties of DNA molecules, supercoiling, knotting, and catenation, are intimately connected with essential biological processes, such as gene expression, replication, recombination, and chromosome segregation. Non-trivial DNA topologies present challenges to the molecular machines that process and maintain genomic information, for example, by creating unwanted DNA entanglements. At the same time, topological distortion can facilitate DNA-sequence recognition through localized duplex unwinding and longer-range loop-mediated interactions between the DNA sequences. Topoisomerases are a special class of essential enzymes that homeostatically manage DNA topology through the passage of DNA strands. The activities of these enzymes are generally investigated using circular DNA as a model system, in which case it is possible to directly assay the formation and relaxation of DNA supercoils and the formation/resolution of knots and catenanes. Some topoisomerases use ATP as an energy cofactor, whereas others act in an ATP-independent manner. The free energy of ATP hydrolysis can be used to drive negative and positive supercoiling or to specifically relax DNA topologies to levels below those that are expected at thermodynamic equilibrium. The latter activity, which is known as topology simplification, is thus far exclusively associated with type-II topoisomerases and it can be understood through insight into the detailed non-equilibrium behavior of type-II enzymes. We use a non-equilibrium topological-network approach, which stands in contrast to the equilibrium models that are conventionally used in the DNA-topology field, to gain insights into the rates that govern individual transitions between topological states. We anticipate that our quantitative approach will stimulate experimental work and the theoretical/computational modeling of topoisomerases and similar enzyme systems.

Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination.

In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of loxP DNA target sites. The enzyme system's strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral, planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. We use the kinetics of loop closure involving closely spaced (131-151 bp) loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-of-plane HJ distortion, which unequivocally stands in contrast to the square-planar intermediate geometry from Cre-loxP crystal structures and those of other int-superfamily recombinases. J measurements for an HJ-isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex is consistent with temporal averaging of right-handed and achiral structures. Our approach connects the static pictures provided by crystal structures and the natural dynamics of macromolecules in solution, thus advancing a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms.

Deconvolution of nucleic-acid length distributions: a gel electrophoresis analysis tool and applications.

Next-generation DNA-sequencing (NGS) technologies, which are designed to streamline the acquisition of massive amounts of sequencing data, are nonetheless dependent on various preparative steps to generate DNA fragments of required concentration, purity and average size (molecular weight). Current automated electrophoresis systems for DNA- and RNA-sample quality control, such as Agilent's Bioanalyzer® and TapeStation® products, are costly to acquire and use; they also provide limited information for samples having broad size distributions. Here, we describe a software tool that helps determine the size distribution of DNA fragments in an NGS library, or other DNA sample, based on gel-electrophoretic line profiles. The software, developed as an ImageJ plug-in, allows for straightforward processing of gel images, including lane selection and fitting of univariate functions to intensity distributions. The user selects the option of fitting either discrete profiles in cases where discrete gel bands are visible or continuous profiles, having multiple bands buried under a single broad peak. The method requires only modest imaging capabilities and is a cost-effective, rigorous alternative characterization method to augment existing techniques for library quality control.

Kinetic pathways of topology simplification by Type-II topoisomerases in knotted supercoiled DNA.

The topological state of covalently closed, double-stranded DNA is defined by the knot type $K$ and the linking-number difference $\Delta Lk$ relative to unknotted relaxed DNA. DNA topoisomerases are essential enzymes that control the topology of DNA in all cells. In particular, type-II topoisomerases change both $K$ and $\Delta Lk$ by a duplex-strand-passage mechanism and have been shown to simplify the topology of DNA to levels below thermal equilibrium at the expense of ATP hydrolysis. It remains a key question how small enzymes are able to preferentially select strand passages that result in topology simplification in much larger DNA molecules. Using numerical simulations, we consider the non-equilibrium dynamics of transitions between topological states $(K,\Delta Lk)$ in DNA induced by type-II topoisomerases. For a biological process that delivers DNA molecules in a given topological state $(K,\Delta Lk)$ at a constant rate we fully characterize the pathways of topology simplification by type-II topoisomerases in terms of stationary probability distributions and probability currents on the network of topological states $(K,\Delta Lk)$. In particular, we observe that type-II topoisomerase activity is significantly enhanced in DNA molecules that maintain a supercoiled state with constant torsional tension. This is relevant for bacterial cells in which torsional tension is maintained by enzyme-dependent homeostatic mechanisms such as DNA-gyrase activity.

Towards automatic image analysis and assessment of the multicellular apoptosis process.

Apoptotic programmed cell death (PCD) is a fundamental aspect of developmental maturation. However, the authors' understanding of apoptosis, especially in the multi-cell regime, is incomplete because of the difficulty of identifying dying cells by conventional strategies. Real-time in vivo microscopy of Drosophila, an excellent model system for studying the PCD during development, has been used to uncover plausible collective apoptosis at the tissue level, although the dynamic regulation of the process remains to be deciphered. In this work, the authors have developed an image-analysis program that can quantitatively analyse time-lapse microscopy of live tissues undergoing apoptosis with a fluorescent nuclear marker, and subsequently extract the spatiotemporal patterns of multicellular response. The program can process a large number of cells (>103) automatically tracked across sets of image frames. It is applied to characterise the apoptosis of Drosophila wing epithelium at eclosion. Using the natural anatomic structures as reference, the authors identify dynamic patterns in the progression of PCD within the Drosophila tissues. The results not only confirm the previously observed collective multi-cell behaviour from a quantitative perspective, but also reveal a plausible role played by the anatomic structures, such as the wing veins, in the PCD propagation across the Drosophila wing.

A mathematical model for apoptotic switch in Drosophila.

Apoptosis is an evolutionarily-conserved process of autonomous cell death. The molecular switch mechanism underlying the fate decision of apoptosis in mammalian cells has been intensively studied by mathematical modeling. In contrast, the apoptotic switch in invertebrates, with highly conserved signaling proteins and pathway, remains poorly understood mechanistically and calls for theoretical elucidation. In this study, we develop a mathematical model of the apoptosis pathway in Drosophila and compare the switch mechanism to that in mammals. Enumeration of the elementary reactions for the model demonstrates that the molecular interactions among the signaling components are considerably different from their mammalian counterparts. A notable distinction in network organization is that the direct positive feedback from the effector caspase (EC) to the initiator caspase in mammalian pathway is replaced by a double-negative regulation in Drosophila. The model is calibrated by experimental input-output relationship and the simulated trajectories exhibit all-or-none bimodal behavior. Bifurcation diagrams confirm that the model of Drosophila apoptotic switch possesses bistability, a well-recognized feature for an apoptosis system. Since the apoptotic protease activating factor-1 (APAF1) induced irreversible activation of caspase is an essential and beneficial property for the mammalian apoptotic switch, we perform analysis of the bistable caspase activation with respect to the input of DARK protein, the Drosophila homolog of APAF1. Interestingly, this bistable behavior in Drosophila is predicted to be reversible. Further analysis suggests that the mechanism underlying the systems property of reversibility is the double-negative feedback from the EC to the initiator caspase. Using theoretical modeling, our study proposes plausible evolution of the switch mechanism for apoptosis between organisms.

Automatic image analysis of multicellular apoptosis process.

Apoptotic programmed cell death (PCD) is a common and fundamental aspect of developmental maturation. Image processing techniques have been developed to detect apoptosis at the single-cell level in a single still image, while an efficient algorithm to automatically analyze the temporal progression of apoptosis in a large population of cells is unavailable. In this work, we have developed an ImageJ-based program that can quantitatively analyze time-lapse microscopy movies of live tissues undergoing apoptosis with a fluorescent cellular marker, and subsequently extract the temporospatial pattern of multicellular response. The protocol is applied to characterize apoptosis of Drosophila wing epithelium cells at eclosion. Using natural anatomic structures as reference, we identify dynamic patterns in the progression of apoptosis within the wing tissue, which not only confirms the previously observed collective cell behavior from a quantitative perspective for the first time, but also reveals a plausible role played by the anatomic structures in Drosophila apoptosis.