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Rep‐X can unwind thousands of base pairs processively even against large forces. To probe the unwinding mechanism, we developed a conformational control assay which turned wild type Rep monomers with no detectable DNA unwinding activity into super‐helicases (Rep‐X) via internal covalent crosslinking. Second, we tested the effects of the DNA lesions on Rep translocation, observing that the irregularities encompassing 1‐3 nucleotides in the backbone and nucleobases only caused transient stalls. First, we perturbed the ssDNA binding network via biochemical mutations that led to determination of the key residues that control shuttling speed, ATPase activity, and directionality. Using the repetitive shuttling assay, we explored the translocation mechanism of Rep in detail. We concluded that the repetitions are induced by the lack of ssDNA track ahead of the Rep. We investigated the mechanism of repetitive shuttling and discovered the same behavior on various DNA substrates. When a translocating Rep on a 3’‐overhang DNA reaches the DNA duplex junction, instead of dissociating, it snaps back to the 3’ end, restarting another shuttling cycle. We used single molecule Förster resonance energy transfer (FRET) and optical tweezers force spectroscopy to study translocation and unwinding mechanism of Escherichia coli Rep, a model helicase with 3’5’ motor translocation activity on single‐stranded DNA (ssDNA) fueled by ATP hydrolysis.

Helicases form a ubiquitous class of enzymes that function in many aspects of nucleic acid metabolism which is central to life. Degree Level Dissertation Keyword(s)Ībstract In the last 15 years, single molecule techniques have allowed us to observe the transient, heterogeneous, and multi‐step behavior of biomolecules often averaged out and lost in ensemble assays. Committee Member(s)ĭepartment of Study Physics Discipline Physics Degree Granting Institution University of Illinois at Urbana-Champaign Degree Name Ph.D.

Title Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins Author(s) Arslan, Sinan Date of Publication Director of Research (if dissertation) or Advisor (if thesis) Ha, Taekjip Doctoral Committee Chair(s) Selvin, Paul R.