Single-molecule optical tweezers studies of the mechanism of DNA unwinding by a model helicase

Helicases are enzymes that use ATP hydrolysis to translocate on single-stranded nucleic acids and unwind double-stranded nucleic acid duplexes in a stepwise manner. Understanding the detailed mechanism by which helicases unwind DNA and RNA requires precise measurement of fundamental parameters such as the step size, or number of base pairs unwound for each cycle of motion along the nucleic acid substrate. UvrD is a prototypical helicase from E. coli that translocates 3’ to 5’ on DNA and is involved in numerous DNA repair pathways, such as methyl-directed mismatch repair. In mismatch repair, the helicase unwinding ability of UvrD is greatly enhanced by interaction with accessory protein MutL. Despite its status as a well-characterized model system, there are still aspects of the unwinding mechanism of UvrD that are not completely understood. A consensus on its step size is lacking, and all previous step size measurements for UvrD have been indirect. In addition, the difference in stepping behavior between monomeric and dimeric UvrD, the two major oligomeric forms of the helicase, is not clear. In a larger biological context, there are also elements of the mechanism of activation of UvrD helicase by partner protein MutL during mismatch repair that are unclear. In this dissertation, we use high-resolution single-molecule optical tweezers to dissect the fundamental mechanism by which UvrD unwinds DNA. Our main focus is the base pair level stepping behavior of UvrD by itself, but we also study the mechanism of activation of UvrD by MutL. First, we directly measure the stepping behavior of monomeric UvrD as it unwinds a DNA hairpin. We measure an average step size of 3 base pairs (bp) independent of ATP, force, or DNA sequence. The step size is highly variable and further analysis of the step size distribution reveals multiple peaks below the average of 3 bp and a periodicity of ~0.5 bp, suggesting that 3 bp is not the elemental stepping unit. Analysis of dwell time kinetics across all ATP reveals that larger steps are preceded by longer dwell times and a higher number of ATP binding events. The data support a mechanism where UvrD unwinds 1 bp per each ATP hydrolyzed but sequesters the nascent ssDNA as loops that are released after multiple rounds of unwinding. This model is further supported by molecular dynamics simulations that identify several basic amino acid residues within the motor core of the protein that interact with a high probability with the ssDNA backbone to form loops. Structural and sequence alignment data suggest that this stepping mechanism may be conserved among other closely related helicases. Next, we probe the stepping dynamics of dimeric UvrD. We find that nearly all major features of the data for monomer unwinding, including the 3 bp average steps size, large step size variability and prevalence of non-integer steps, and kinetic analysis suggesting a strong correlation between dwell time and the size of the succeeding step, also apply to UvrD dimers. We believe that a similar strand sequestration mechanism may be at play for dimeric UvrD unwinding, although it is not possible to discern which individual subunits of the dimer are involved in strand sequestration from the present data. Last, we turn our attention to the mechanism of UvrD helicase activity enhancement by partner protein MutL. Using a hairpin DNA substrate, we find that MutL moderately increases the processivity, or average amount of DNA unwound per unwinding attempt, for monomeric UvrD from 15 to 25 bp. Using a gapped DNA substrate to monitor longer distance unwinding, we observe that MutL modestly increases the average processivity of UvrD at these conditions by facilitating rare events where longer regions of DNA (> 500 bp) are unwound. Importantly, these long-distance unwinding events only happen when both MutL is present and multiple UvrD can continually bind to, dissociate from, and rebind the DNA. These results suggest that MutL can increase the processivity of a UvrD monomer but must continually load multiple UvrD from solution onto the DNA to enable unwinding of longer, kilobase level distances. Further experiments will be needed to test this hypothesis more rigorously.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Carney, Sean P

Contributors dc:contributor

• Chemla, Yann R
• Gruebele, Martin
• Selvin, Paul R
• Murphy, Catherine J

• Optical Tweezers
• Helicases
• Single-molecule
• Dna
• Nucleic Acids
• Dna-binding Proteins
• Molecular Modelling

Statement dc:rights

• Copyright 2023 Sean Carney

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/120201

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F120201