Graduate Thesis Or Dissertation

 

Single-Molecule Investigations of Enzyme Dynamics: RecBCD Helicase Motion and glmS Ribozyme Cleavage Public Deposited

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https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/v405s9425
Abstract
  • Enzyme dynamics are a critical factor in enzyme function. Probing individual enzymes and enzymatic systems elucidates the mechanistic basis of their dynamics. Here, with two distinct single-molecule, biophysical approaches, we examine two particular enzymatic systems. RecBCD is a multifunctional enzyme that repairs double-stranded DNA breaks and degrades unwanted DNA in Escherichia coli. To probe how this superfamily-1 helicase couples ATP binding and hydrolysis to unwind and directionally translocate along double-stranded DNA, we used optical tweezers featuring 1-base-pair resolution (0.1-2.3 Hz, F = 6 pN) to directly measure interactions between single RecBCD helicases and their DNA substrate. Surprisingly, the RecBCD-DNA complex exhibited large, multi-base-pair conformational dynamics (4.4 +/- 0.3 bp, mean +/- S.E.M., 0.1-10 Hz) that were present both between unwinding steps and in the complete absence of ATP. To understand the origin of the conformational dynamics, we made three observations. First, the onset of the conformational dynamics depended on 5'-ssDNA length, which interacts with the RecD motor, and were consistent with the onset of helicase activity, as established in prior biochemical studies. Second, conformational dynamics of RecBCD bound to a cross-linked DNA construct were suppressed, indicating that the RecBCD "pin," which interacts with the junction, undergoes rapid back-and-forth translocations along the DNA. Third, conformational dynamics were reduced in the presence of nonhydrolyzable ATP analogs. These results indicate that when RecD is engaged RecBCD actively destabilizes the duplex, and that these conformational dynamics are modulated by the nucleotide-bound state of the enzyme. These studies signify the first real-time measurements of previously-unseen destabilization dynamics of the DNA duplex by RecBCD and provide insight into the unwinding mechanism of superfamily-1 helicases. In the effort to develop highly-sensitive biosensors for medical diagnostics, environmental monitoring, and industrial quality control, nucleic-acid-based biosensors offer a number of competitive advantages over antibodies for sensing applications. We explored a naturally-occurring biosensor, the glmS ribozyme, a RNA molecule that has the remarkable ability to recognize a specific metabolite, catalyze self-cleavage upon its binding, and regulate gene expression. We developed a new, single-molecule assay to detect individual glmS ribozyme cleavage events with video microscopy that can be generalized to other nucleic-acid-based biosensors.
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  • 2011
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  • 2019-11-15
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