All motors have to decide is what to do with the DNA that is given them

This is the published version. Copyright 2014 De Gruyter.DNA translocases are a diverse group of molecular motors responsible for a wide variety of cellular functions. The goal of this review is to identify common aspects in the mechanisms for how these enzymes couple the binding and hydrolysis of ATP to their movement along DNA. Not surprisingly, the shared structural components contained within the catalytic domains of several of these motors appear to give rise to common aspects of DNA translocation. Perhaps more interesting, however, are the differences between the families of translocases and the potential associated implications both for the functions of the members of these families and for the evolution of these families. However, as there are few translocases for which complete characterizations of the mechanisms of DNA binding, DNA translocation, and DNA-stimulated ATPase have been completed, it is difficult to form many inferences. We therefore hope that this review motivates the necessary further experimentation required for broader comparisons and conclusions

Kinetic Mechanism for Single stranded DNA binding and Translocation by S. cerevisiae Isw2

The chromatin remodeling complex Isw2 from S. cerevisiae (yIsw2) mobilizes nucleosomes through an ATP-dependent reaction that is coupled to the translocation of the helicase domain of the enzyme along intranucleosomal DNA. In this study we demonstrate that yIsw2 is capable of translocating along single-stranded DNA in a reaction that is coupled to ATP hydrolysis. We propose that single-stranded DNA translocation by yIsw2 occurs through a series of repeating uniform steps with an overall macroscopic processivity of P = (0.90 ± 0.02), corresponding to an average translocation distance of (20 ± 2) nucleotides before dissociation. This processivity corresponds well to the processivity of nucleosome sliding by yIsw2 thus arguing that single-stranded DNA translocation or tracking may be fundamental to the double-stranded DNA translocation required for effective nucleosome mobilization. Furthermore, we find evidence that a slow initiation process, following DNA binding, may be required to make yIsw2 competent for DNA translocation. We also provide evidence that this slow initiation process may correspond to the second step of a two-step DNA binding mechanism by yIsw2 and a quantitative description of the kinetics of this DNA binding mechanism

Ensemble Methods for Monitoring Enzyme Translocation along Single Stranded Nucleic Acids

We review transient kinetic methods developed to study the mechanism of translocation of nucleic acid motor proteins. One useful stopped-flow fluorescence method monitors arrival of the translocase at the end of a fluorescently labeled nucleic acid. When conducted under single-round conditions the time courses can be analyzed quantitatively using n-step sequential models to determine the kinetic parameters for translocation (rate, kinetic step size and processivity). The assay and analysis discussed here can be used to study enzyme translocation along a linear lattice such as ssDNA or ssRNA. We outline the methods for experimental design and two approaches, along with their limitations, that can be used to analyze the time courses. Analysis of the full time courses using n-step sequential models always yields an accurate estimate of the translocation rate. An alternative semi-quantitative “time to peak” analysis yields accurate estimates of translocation rates only if the enzyme initiates translocation from a unique site on the nucleic acid. However, if initiation occurs at random sites along the nucleic acid, then the “time to peak” analysis can yield inaccurate estimates of even the rates of translocation depending on the values of other kinetic parameters, especially the rate of dissociation of the translocase. Thus, in those cases analysis of the full time course is needed to obtain accurate estimates of translocation rates

Single stranded DNA translocation of E. coli UvrD monomer is tightly coupled to ATP hydrolysis

E. coli UvrD is an SF1A helicase/translocase that functions in several DNA repair pathways. A UvrD monomer is a rapid and processive single-stranded (ss) DNA translocase, but is unable to unwind DNA processively in vitro. Based on data at saturating ATP (500 μM) we proposed a non-uniform stepping mechanism in which a UvrD monomer translocates with biased (3′ to 5′) directionality while hydrolyzing 1 ATP per DNA base translocated, but with a kinetic step-size of 4–5 nucleotides/step, suggesting a pause occurs every 4–5 nucleotides translocated. To further test this mechanism we examined UvrD translocation over a range of lower ATP concentrations (10–500 μM ATP), using transient kinetic approaches. We find a constant ATP coupling stoichiometry of ~1 ATP/DNA base translocated even at the lowest ATP concentration examined (10 μM) indicating that ATP hydrolysis is tightly coupled to forward translocation of a UvrD monomer along ssDNA with little slippage or futile ATP hydrolysis during translocation. The translocation kinetic step size remains constant at 4–5 nucleotides/step down to 50 μM ATP, but increases to ~7 nucleotides/step at 10 μM ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP, but with little futile ATP hydrolysis

Liquid simulation with mesh-based surface tracking

Animating detailed liquid surfaces has always been a challenge for computer graphics researchers and visual effects artists. Over the past few years, researchers in this field have focused on mesh-based surface tracking to synthesize extremely detailed liquid surfaces as efficiently as possible. This course provides a solid understanding of the steps required to create a fluid simulator with a mesh-based liquid surface. The course begins with an overview of several existing liquid-surface-tracking techniques and the pros and cons of each method. Then it explains how to embed a triangle mesh into a finite-difference-based fluid simulator and describes several methods for allowing the liquid surface to merge together or break apart. The final section showcases the benefits and further applications of a mesh-based liquid surface, highlighting state-of-the-art methods for tracking colors and textures, maintaining liquid volume, preserving small surface features, and simulating realistic surface-tension waves

Proteins are Not Recruited: A Plea for Better Diction

Nearly all biological processes proceed or are controlled by protein-protein or protein-ligand binding reactions. Using anthropomorphic language to describe these interactions conveys an incorrect physical description of these processes while simultaneously minimizing the importance of the thermodynamics underpinning the associated interactions. Indeed, we should never say that proteins are recruited to binding partners or binding sites since this implies both a non- existent level of communication within biological systems and a non-existent process by which proteins or binding sites actively seek other proteins. Both of these fictions hinder our ability to determine quantitatively or qualitatively distinct biophysical descriptions of the associated systems. Here we present examples of how interactions typically described as protein recruitment can be more accurately and often more simply described as variations within binding equilibria. We argue that this approach is better for describing protein-protein and protein-ligand binding, even when the objective is only a qualitative description, especially for discussions with students in courses and research groups as it provides testable models for these interaction

Kinetic Mechanism of DNA Translocation by the RSC Molecular Motor

ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation of these enzymes along the nucleosomal DNA. Using a fluorescence stopped-flow assay we monitored DNA translocation by a minimal RSC motor and through global analysis of these time courses we have determined that this motor has a macroscopic translocation rate of 2.9 bp/s with a step size of 1.24 bp. From the complementary quantitative analysis of the associated time courses of ATP consumption during DNA translocation we have determined that this motor has an efficiency of 3.0 ATP/bp, which is slightly less that the efficiency observed for several genetically related DNA helicases and which likely results from random pausing by the motor during translocation. Nevertheless, this motor is able to exert enough force during translocation to displace streptavidin from biotinylated DNA. Taken together these results are the necessary first step for quantifying both the role of DNA translocation in nucleosome repositioning by RSC and the efficiency at which RSC couples ATP binding and hydrolysis to nucleosome repositioning

Calculus-enhanced energy-first curriculum for introductory physics improves student performance locally and in downstream courses

Here we demonstrate the benefits of a new curriculum for introductory calculus-based physics that motivates classical mechanics using a modified version of Hamiltonian mechanics. This curriculum shifts the initial focus of instruction away from forces and the associated vector mathematics, which are known to be problematic for students, to the scalar quantity energy, which is more closely aligned with their previously established intuition, and associated differential and integral calculus. We show that implementation of this calculus-enhanced “energy-first” curriculum resulted in higher normalized gains on the Force Concept Inventory exam for all students and improved performance in downstream engineering courses for students with lower ACT math scores. In other words, the downstream benefits were largest for students with lower math abilities who also pose a larger retention risk. This new curriculum thus has the potential to improve student retention by specifically helping the students who need help the most, including traditionally underserved populations who often have weaker mathematics preparation. We propose future work to investigate whether this new curriculum has lowered the math transference barrier to learning in introductory physics, resulting concomitantly in improvements in student learning of classical mechanics and in student fluency with applied mathematics.Northrop Grumman Foundatio