Person: Harada, Bryan T.
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Harada
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Bryan T.
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Harada, Bryan T.
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Publication Histone H4 tail mediates allosteric regulation of nucleosome remodelling by linker DNA(2014) Hwang, William; Deindl, Sebastian; Harada, Bryan T.; Zhuang, XiaoweiISWI-family remodelling enzymes regulate access to genomic DNA by mobilizing nucleosomes1. These ATP-dependent chromatin remodellers promote heterochromatin formation and transcriptional silencing1 by generating regularly-spaced nucleosome arrays2-5. The nucleosome-spacing activity arises from regulation of nucleosome translocation by the length of extranucleosomal linker DNA6-10, but the underlying mechanism remains unclear. Here, we studied nucleosome remodelling by human ACF, an ISWI enzyme comprised of a catalytic subunit, Snf2h, and an accessory subunit, Acf12,11-13. We found that ACF senses linker DNA length through an interplay between its accessory and catalytic subunits mediated by the histone H4 tail of the nucleosome. Mutation of AutoN, an auto-inhibitory domain within Snf2h that bears sequence homology to the H4 tail14, abolished the linker-length sensitivity in remodelling. Addition of exogenous H4-tail peptide or deletion of the nucleosomal H4 tail also diminished the linker-length sensitivity. Moreover, the accessory subunit Acf1 bound the H4-tail peptide and DNA in a manner that depended on its N-terminal domain, and lengthening the linker DNA in the nucleosome reduced the proximity between Acf1 and the H4 tail. Deletion of the N-terminal portion of Acf1 (or its homologue in yeast) abolished linker-length sensitivity in nucleosome remodeling and led to severe growth defects in vivo. Taken together, our results suggest a mechanism for nucleosome spacing where linker DNA sensing by Acf1 is allosterically transmitted to Snf2h through the H4 tail of the nucleosome. For nucleosomes with short linker DNA, Acf1 preferentially binds to the H4 tail, allowing AutoN to inhibit the ATPase activity of Snf2h. As the linker DNA lengthens, Acf1 shifts its binding preference to the linker DNA, freeing the H4 tail to compete AutoN off the ATPase and thereby activating ACF.Publication Single Molecule FRET Studies of Reverse Transcription and Chromatin Remodeling(2015-08-21) Harada, Bryan T.; Kadoch, Cigall; Loparo, Joseph J.; Yusufzai, Timur; Hogle, James M.The measurement of Förster resonance energy transfer (FRET) at the single-molecule level provides a powerful method for monitoring the structural dynamics of biomolecular systems in real time. These single-molecule FRET (smFRET) assays enable the characterization of the transient intermediates that form during enzymatic processes, providing information about the mechanism and regulation of the enzymes involved. In this dissertation, I develop and use smFRET assays to study two processes driven by motor proteins—reverse transcription and chromatin remodeling—and reveal novel features of their mechanism and regulation. Reverse transcription of the human immunodeficiency virus genome initiates from a cellular tRNA primer that is bound to a specific sequence on the viral RNA (vRNA). During initiation, reverse transcriptase (RT) exhibits a slow mode of synthesis characterized by pauses at specific locations, and RT transitions to a faster mode of synthesis after the extension of the tRNA primer by six nucleotides. By using smFRET to examine how RT interacts with the tRNA-vRNA substrate, we found that RT binds to its substrate in either an active or inactive orientation and samples the two orientations during a single binding event. The equilibrium between these two orientations is a major factor influencing the activity and pausing of the enzyme, and a specific RNA secondary structure in the vRNA substrate modulates the binding mode of RT, determining the locations of the pauses and the transition to the faster mode of synthesis. These results provide a mechanistic explanation for the changes in RT activity observed during initiation and show how the dynamics of a ribonucleoprotein complex can regulate enzymatic activity. ISWI family chromatin remodelers are another family of motor enzymes regulated by nucleic acid structures. These enzymes are involved in creating evenly spaced nucleosome arrays, and this nucleosome spacing activity arises from the regulation of the enzymes’ catalytic activity by the amount of linker DNA present on the nucleosome. We use smFRET and other biochemical assays to monitor intermediates of the remodeling reaction and examine various remodeler mutants in order to elucidate the mechanism of this regulation. These experiments led to the discovery of an allosteric mechanism by which one subunit of the ISWI remodeling complex communicates the presence of linker DNA to the the catalytic subunit by modulating the availability of the histone H4 tail. These results provide a mechanistic explanation for the nucleosome spacing activity of the ISWI chromatin remodelers. Like the ISWI chromatin remodelers, the SWI/SNF family chromatin remodelers can also reposition nucleosomes, but they may do so by a different mechanism. To investigate the mechanism by which these remodelers move DNA around the nucleosome, we used smFRET to monitor the structural dynamics of nucleosomes during remodeling by the SWI/SNF enzymes. Our results are consistent with movement of the DNA along its canonical path without substantial lifting of DNA off the edges of the nucleosome or displacement of the H2A-H2B dimer. We observe DNA translocation in 1-2 bp increments at both edges of the nucleosome, which suggests that the motion of DNA at the edges of the nucleosome is driven directly by the action of the ATPase near the dyad of the nucleosome.