High Temporal Resolution DNA-Flap Endonuclease 1 Interaction at the Single Molecule Level

  • Paul David Harris

Student thesis: Doctoral Thesis

Abstract

Numerous short flapped DNA structures are created during the semi-discontinuous replication. These toxic intermediates are quickly resolved to produce a fully intact duplex of replicated DNA. Structure specific nuclease are key to resolving these structures, and show a high degree of specificity for their cognate substrate structures while being essentially insensitive to nucleotide sequence. Herein I demonstrate through confocal based single molecule experiments that the 5’ structure specific nuclease Flap Endonuclease 1 (FEN1) achieves its substrate specificity by coupling the bending of DNA substrate with structuring of the active site in a way that non-cognate structures binding is significantly destabilized and enzymatic features are incapable of structuring in the absence of particular substrate features, in particular a single nucleotide 3’ flap the FEN1 induces in nearly all DNA substrates. Debate remained over whether DNA was bound via a conformational capture or induced fit mechanism, and so I proceed to investigate the dynamics of the DNA itself in solution. Conclusions about conformational capture or induced fit remain elusive, however I did determine that DNA structures are rigidified by charge repulsion, an effect lessened by the salt concentration, which functions to shield the negative charge of DNA from itself. Additionally unstacking of the DNA in nicked structures incurs a significant free energy penalty, which FEN1 overcomes by its hydrophobic wedge motif, lending credence to an induced fit mechanism.
Date of AwardJul 2020
Original languageEnglish (US)
Awarding Institution
  • Biological, Environmental Science and Engineering
SupervisorSatoshi Habuchi (Supervisor)

Keywords

  • Single Molecule
  • FEN1
  • DNA dynamics
  • unstacking
  • FRET
  • induced fit

Cite this

'