This thesis explores two topics. The first is shotgun DNA mapping (SDM). Ability to map polymerases and nucleosomes on chromatin is important for understanding the impact of chromatin remodeling on key cellular processes. Current methods have produced a wealth of information that demonstrates this importance, but key information is elusive in these methods. We are pursuing a new single-molecule chromatin mapping method based on unzipping native chromatin molecules with optical tweezers. The first step we are taking towards this ability is SDM. This is the ability to identify the genomic location of a random DNA fragment based on its naked DNA unzipping forces compared with simulated unzipping forces of a published genome. We show that ∼32 separate experimental unzipping curves for pBR322 were correctly matched to their simulated unzipping curves hidden in a background of the ∼2700 sequences neighboring XhoI sites in the S. cerevisiae (yeast) genome. We describe this method and characterize its robustness as well as discuss future applications.
The second topic is a discrete state model for kinesin-1's processivity. Kinesin-1 is a homodimeric molecular motor protein that uses ATP and a hand-over-hand motion to transport cargo along microtubules. Minimal kinetic models are often developed to both explain kinesin's hand-over-hand forward-stepping behavior and to infer important kinetic rate constants from experimental data. These minimal models are often limited to a handful of two-headed states on a core cycle. However, it is not always clear how to evolve these core-cycle models to explain more complex behavior. We have developed a kinetic model without a pre-defined core cycle. Our model includes 80 two-headed states and permits transitions between any two states that differ by a single catalytic or binding event. We constrain the rate constants as much as possible by published rates and mechanical strain in the kinesin neck linkers and their docking state. We present a model for neck-linker modulation of head and nucleotide binding and unbinding rates. We show that our model reproduces generally-accepted experimental results. The core cycles that emerge are slightly different than those seen in previous experiments. We also explore how processivity and speed change with neck linker length.
|Advisor:||Koch, Steven J.|
|Commitee:||Atlas, Susan R., Dunlap, David H., Fields, Douglas E., Koch, Steven J.|
|School:||The University of New Mexico|
|School Location:||United States -- New Mexico|
|Source:||DAI-B 72/04, Dissertation Abstracts International|
|Keywords:||DNA unzipping, Kinesin, Microtubules, Worm-like chains|
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