ORCID

http://orcid.org/0000-0002-3852-0322

Date of Award

Winter 12-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Computational & Molecular Biophysics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

I have examined the effects of deleting the nuclease domain of the E. coli helicase RecBCD on the rates of ATP-independent DNA melting, single stranded (ss) DNA translocation, and double stranded (ds) DNA unwinding by RecBCD. The canonical role of the nuclease domain is DNA degradation, but the removal of this domain showed unexpected effects on other RecBCD activities including DNA binding, melting, and unwinding. This thesis presents a mechanistic study of DNA unwinding by RecBCD and a RecBCD variant with the nuclease domain deleted (RecBΔnucCD). I examined the rates of ssDNA translocation and dsDNA unwinding by RecBCD and RecBΔnucCD using fluorescence stopped-flow assays. Deletion of the nuclease domain does not significantly affect the rate of ssDNA translocation in either the 3’ to 5’ or 5’ to 3’ directions but slows the rate of dsDNA unwinding by 25-45%. These results are consistent with a model in which RecBCD unwinds dsDNA through iterations of binding free energy-driven ATP-independent DNA base pair melting and ATP hydrolysis driven ssDNA translocation along the melted base pairs. Slower unwinding by RecBΔnucCD is likely due to an effect on its ability to destabilize or melt the duplex DNA. I also examined the ability of RecBCD and RecBΔnucCD to initiate unwinding from DNA with blunt, partially pre-melted or fully pre-melted ends using fluorescence stopped-flow experiments. I found that RecBCD is able to initiate efficiently from all DNA end types. The rates of unwinding initiation by RecBΔnucCD, however, are up to 10-fold slower than RecBCD and are sensitive to DNA end type. Additionally, deletion of the nuclease domain decreases the fraction of initially productive RecBCD-DNA complexes. These results suggest DNA melting is necessary for efficient initiation of DNA unwinding, and the slow initiation by RecBΔnucCD emphasizes that the deletion of the nuclease domain decreases DNA melting by RecBCD. I also present preliminary single molecule total internal reflection fluorescence experiments designed to directly examine the extent and dynamics of ATP-independent DNA melting by RecBCD and RecBΔnucCD. These results were difficult to analyze and interpret due in large part to Cy3 protein induced fluorescence enhancement (PIFE) upon binding of RecBCD, which is then transferred to Cy5 via FRET. We, therefore, turned to ensemble fluorescence binding experiments to better understand the nature of the fluorescence signals that were observed in the smTIRF experiments. Results of these experiments suggest that using Cy5 and black hole quencher to label DNA may be a viable labeling scheme for measuring DNA melting by RecBCD. Additionally, careful fluorophore placement and fluorescence calibrations and corrections will be necessary to measure DNA melting. Combined, my results indicate that the nuclease domain has a role in regulating dsDNA unwinding and initiation, possibly through DNA melting and allosteric interactions within RecBCD.

Language

English (en)

Chair and Committee

Timothy M. Lohman

Committee Members

Eric Galburt

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