Kinesin-14s are microtubule-based motor proteins that play important roles in cell division. They were originally thought to be minus-end-directed nonprocessive motors that exhibit directional preference toward the microtubule minus ends in multi-motor ensembles but are unable to generate processive (continuous) motility on single microtubules as individual motors. During the past five years, we and others have discovered several "unconventional" kinesin-14 motors that all contain the ability to generate processive motility as individual motors on single microtubules. In this talk, I will present a series of unexpected yet exciting findings from my lab that have markedly expanded current view of the design and operation principles of kinesin-14 motors.

Cre recombinase catalyzes site-specific recombination of DNA. This well-studied member of the Tyrosine recombinase family of enzymes does not require any co-factors or downstream repair mechanisms to bind DNA at loxP recognition sequences and cause strand exchange, insertion or deletion. For these reasons, Cre recombinase has been widely used as a gene-editing tool. At the start of the reaction mechanism, two Cre molecules bind to a loxP site and further assemble to form a tetrameric synaptic complex Cre₄-loxP₂. While previous single molecule and ensemble kinetics studies implicated protein conformational changes (i.e., dynamics) in the progression of the Cre-loxP recombination pathway, the motions, timescales and their structural basis remained poorly understood. We aimed to characterize the dynamics of Cre recombinase at atomic resolution using solution NMR spectroscopy. Backbone ¹⁵N T₁, T₂ and {¹H}-¹⁵N hetNOE relaxation measurements on the catalytic domain of Cre in the absence and presence of DNA substrate identified significant regions that exhibit dynamics on the ps-ns timescales. Our findings provide evidence for the significance of protein dynamics in the progression of Cre-loxP reaction mechanism and give strong insight into the hitherto unknown solution structure of free Cre recombinase. Furthermore, through truncation mutagenesis, we identified the significance of the C-terminal alpha helix in Cre's "fly-casting" mechanism for formation of synaptic complexes. As expected, our preliminary solution structure of free Cre catalytic domain shows significant changes from crystal structure of Cre in synaptic complexes, indicating that site-specific DNA binding acts as a Cre recombinase conformational switch.

Packaging the eukaryotic genome into nucleosomes greatly limits transcription factor (TF) occupancy by restricting binding site accessibility and accelerating TF off-rates. Because of this, nucleosomes are precisely positioned to regulate TF activation of transcription. Reb1 is a TF from S. cerevisiae that can recruit chromatin remodelers and generate nucleosome-depleted regions. Previous studies also suggest that Reb1 preferentially targets nucleosomes at the DNA entry/exit region. To probe Reb1-nucleosome interactions, we inserted a Reb1 binding site in increments of 5 bp throughout the entry/exit region and monitored Reb1 binding and Reb1-induced nucleosome structural changes. Gel shift measurements reveal Reb1 binds nucleosomes with a similar affinity to naked DNA, which is in stark contrast to other TFs such as Gal4 that binds nucleosomal binding sites with drastically lower affinities. This property is shared with eukaryotic pioneer factors Sox2 and Oct4 suggesting Reb1 functions as a pioneer factor. Ensemble FRET measurements show that Reb1 binding to its site within the entry-exit region traps nucleosomes in a partially unwrapped state without evicting histones. This indicates that Reb1 functions to expose nucleosomal DNA and reduce nucleosome stability. Single molecule fluorescence measurements show that despite similar equilibrium affinities, exchange kinetics are 50-fold slower at nucleosomal DNA sites relative to naked DNA, highlighting another distinct nucleosome binding behavior as compared with other TFs that have dramatically increased exchange kinetics at nucleosomal binding sites. From these results, we propose that Reb1 can function as a pioneer factor that induces nucleosome unwrapping and resides at nucleosomal DNA entry/exit sites for minutes to facilitate the recruitment of chromatin remodelers.