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

Disrupted Pathways: Generating Tunable Macromolecular Assembly Pathways

What follows is a pathway; a sequence of individual events, which together form a story. Yet it is still only a small part of what has come before. Biological structures also have individual stories; each composed of simple events in sequence. One story does not tell the whole, for that we must observe many stories, sample them if you will. Together, they bring understanding. Assembly is an emergent property of many individual binding events. Through this, all of the structures that make up life are created. Understanding the regime of possibilities provides insight into both the breadth and tendencies of the system. Cells contain numerous types of individual proteins many of which come together to form larger complexes. I will begin by introducing the elementary building blocks of those protein complexes. An introductory example will provide the first perspective, it will form common ground and allow the telling of the larger story with a shared perspective. Then a case study, a real biological complex and how understanding the progression of its pathways provided insight into the states which it reached. With the elementary operations described, I will move on to laying out the landscape of possible pathways; first for a specific case and then the structure of the assembly pathways themselves. Thus, providing a novel framework for the understanding of the stochastic space of protein complex assembly. Finally, I will provide an example of how making changes in the possible assembly pathways leads to non-intuitive changes in the conclusion of the protein complexes’ stories

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

Effects of nucleosome stability on remodeler-catalyzed repositioning

Chromatin remodelers are molecular motors that play essential roles in the regulation of nucleosome positioning and chromatin accessibility. These machines couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of manipulating chromatin structure through processes that are not completely understood. Here we present a quantitative analysis of nucleosome repositioning by the imitation switch (ISWI) chromatin remodeler and demonstrate that nucleosome stability significantly impacts the observed activity. We show how DNA damage induced changes in the affinity of DNA wrapping within the nucleosome can affect ISWI repositioning activity and demonstrate how assay-dependent limitations can bias studies of nucleosome repositioning. Together, these results also suggest that some of the diversity seen in chromatin remodeler activity can be attributed to the variations in the thermodynamics of interactions between the remodeler, the histones, and the DNA, rather than reflect inherent properties of the remodeler itself

ISWI Remodels Nucleosomes through a Random Walk

The chromatin remodeler ISWI is capable of repositioning clusters of nucleosomes to create well-ordered arrays or moving single nucleosomes from the center of DNA fragments toward the ends without disrupting their integrity. Using standard electrophoresis assays, we have monitored the ISWI-catalyzed repositioning of different nucleosome samples each containing a different length of DNA symmetrically flanking the initially centrally positioned histone octamer. We find that ISWI moves the histone octamer between distinct and thermodynamically stable positions on the DNA according to a random walk mechanism. Through the application of a spectrophotometric assay for nucleosome repositioning, we further characterized the repositioning activity of ISWI using short nucleosome substrates and were able to determine the macroscopic rate of nucleosome repositioning by ISWI. Additionally, quantitative analysis of repositioning experiments performed at various ISWI concentrations revealed that a monomeric ISWI is sufficient to obtain the observed repositioning activity as the presence of a second ISWI bound had no effect on the rate of nucleosome repositioning. We also found that ATP hydrolysis is poorly coupled to nucleosome repositioning, suggesting that DNA translocation by ISWI is not energetically rate-limiting for the repositioning reaction. This is the first calculation of a microscopic ATPase coupling efficiency for nucleosome repositioning and also further supports our conclusion that a second bound ISWI does not contribute to the repositioning reaction

Quantitative Determination of Binding of ISWI to Nucleosomes and DNA Shows Allosteric Regulation of DNA Binding by Nucleotides

The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants: 1/β₁ = 1.3 ± 0.6 nM, and 1/β₂ = 13 ± 7 nM². Furthermore, to improve our understanding of the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogues on substrate binding by ISWI. While the affinity of ISWI for the nucleosome substrate with short lengths of flanking DNA was not affected by the presence of nucleotides, the affinity of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable ATP analogues but not by ADP