Mechanism of cytoskeletal transport and transcription-coupled DNA repair

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PROJECT SUMMARY Cells must organize their contents spatially and temporally. The microtubule cytoskeleton and its associated molecular motors, dynein and kinesin, are the main system used by cells to move cargos, ranging from protein assemblies to entire organelles, including the nucleus. Dynein (~0.5MDa) is a member of the AAA+ family. Active dynein complexes, the only ones capable of transporting cargo, are ~4MDa and consist of two dynein dimers bound to the ~1.0MDa dynactin complex, and an adaptor protein that links them to cargo. Lis1, another essential regulator of dynein, is necessary for their formation. Previously, we showed how Lis1 regulates dynein’s mechanochemistry. Here, we will focus on understanding how the 90kDa Lis1 dimer helps activate and assemble the 4MDa transport complex. Chromatin, with the nucleosome as its basic unit, provides both a solution to the problem of packaging the genome, and a tool to regulate access to it. Among the factors involved in controlling chromatin dynamics are ATP-dependent nucleosome remodelers, which couple ATP hydrolysis to the non-covalent modification of nucleosome structure. Both remodelers and the modifications they catalyze are very diverse, even though all remodelers use the same mechanism, and conserved catalytic core, to break histone-DNA contacts. I am interested in how this common underlying mechanism is modulated to result in the wide array of outcomes of which remodelers are capable. Previously, we focused on model systems representing two of the four families of “canonical” remodelers. Here, we will focus on Rad26 (the yeast ortholog of CSB), an “orphan” remodeler that uses its remodeling-like activity to act on RNA Pol II to help it overcome obstacles or initiate Transcription Coupled DNA Repair (TCR) when the obstacle is a DNA lesion. We aim to understand how Rad26 helps RNA Pol II recognize a lesion from other obstacles and recruit downstream repair factors. We take a structure-guided approach to addressing fundamental mechanistic questions, with cryo- electron microscopy (cryo-EM) as our main technique. We use the structures we generate to formulate mechanistic hypotheses that can be tested, either in house or in collaboration, using a range of techniques including single-molecule biophysics, biochemistry, and cell biology. We have made major contributions to our understanding of the mechanochemical cycle of dynein and its regulation, and to the functional diversity and regulation of nucleosome remodelers. We are also interested in developing tools to solve challenges we encounter along the cryo-EM pipeline and have made important contributions to cryo-EM grid preparation and data processing in the cloud. We are currently developing approaches to increase the efficiency of data collection.