Date of Award

Summer 8-15-2019

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



E. coli UvrD is a superfamily 1A helicase/translocase involved in DNA repair, recombination, and replication. I investigated the role of E. coli MutL, a regulatory protein involved in methyl-directed mismatch DNA repair, in the regulation of UvrD-catalyzed DNA unwinding. Using single molecule fluorescence resonance energy transfer (FRET) and single round stopped-flow DNA unwinding experiments I demonstrated that MutL can activate latent UvrD monomer helicase activity and also stimulate UvrD dimer helicase activity. Furthermore, using analytical ultracentrifugation experiments I determined that a single MutL dimer is sufficient to activate UvrD monomer helicase. DNA unwinding experiments with a series of DNA substrates of varying duplex length under single round unwinding conditions showed that MutL increases the amount of duplex DNA unwound by UvrD in a single binding event. Therefore, MutL acts as a processivity factor by binding to and presumably moving along with UvrD during unwinding. I also showed that MutL requires contacts with the 3’ ssDNA tail for optimal activation of UvrD helicase activity. The C-terminal tail of UvrD is highly variable among SF1A helicases and suggested to interact with MutL, however, the truncated UvrDΔ73 mutant lacking its C-terminal tail is activated by MutL, indicating that the disordered C-terminal domain is not essential for stimulation. I also found that MutL is unable to activate the helicase activity of the structurally similar E. coli Rep helicase, indicating that MutL stimulation is specific to UvrD. Furthermore, MutL also fails to activate the helicase activity of chimeric UvrD containing the 2B sub-domain of Rep helicase. This result demonstrates that MutL activation of the monomeric UvrD helicase is regulated specifically by the 2B sub-domain of UvrD. Using single molecule and ensemble FRET experiments I showed that MutL binding to a UvrD monomer-DNA complex induces partial closing of the 2B sub-domain. Transient kinetic studies of MutL-induced activation of the UvrD helicase and MutL-induced changes in the UvrD 2B sub-domain showed that formation of the partially closed state is on the pathway to forming the active helicase. The kinetic analysis of these two sets of experiments revealed that under the experimentally used MutL concentrations the active MutL-UvrD species are formed predominantly through the conformational selection pathway (>90%) and to a lesser degree through the induced fit pathway (<10%).


English (en)

Chair and Committee

Timothy M. Lohman

Committee Members

Timothy M. Lohman, Peter Burgers, Eric Galburt, Roberto Galletto,


Permanent URL: https://doi.org/10.7936/mps4-eg05