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This thesis focuses on the characterization of DNA polymerases with single-molecule techniques. More specifically, I aimed to study polymerase processivity and fidelity-related conformational changes using assays based on Förster Resonance Energy Transfer (FRET) on a total internal reflection fluorescence (TIRF) microscope.
Chapter 2 reviews some of the recent applications of single-molecule FRET (smFRET) to study DNA and DNA binding proteins, in particular DNA polymerases. The chapter begins with an introduction of FRET, employed to measure distance changes in the 1-10 nm region, and introduces the two most common fluorescence-based implementations of single-molecule techniques: confocal microscopy and TIRF microscopy. The chapter concludes with a short discussion on FRET-based structural modelling, parts of which are applied in practice later in this thesis.
In chapter 3, I report the development of a short, fluorescently labelled DNA sensor to probe DNA polymerization at the single-molecule level. The sensor is a simple primer-template combination labelled with donor and acceptor fluorophores suitable for FRET. The advantage of this assay is that polymerases do not need to be labelled with any fluorophore. I show that the FRET efficiency of the sensors changes significantly upon polymerization of the 25 nucleotide template, and I present time traces showing polymerization of single sensors by three different polymerases (E. coli DNA Polymerase I (KF), human Polymerase Beta (POLB) and the α subunit of bacterial Polymerase III (POLIIIα)). Based on these traces, I can measure polymerase speed and pausing: KF and POLIIIα extended the primer in ~1.0-1.5 s, but POLB was far slower (tens of seconds). I foresee applications for these sensors in the single-molecule field, where they can be used to characterize the processivity of other polymerases, but also for ensemble experiments in which native polymerases need to be tested for activity.
I take a closer look at POLB in chapter 4. This polymerase is involved in DNA repair, and I address the question whether resolving the conformational dynamics of the enzyme can shed new light on fidelity-related mechanisms. Previous work on both KF and POLB showed that the polymerase “fingers” domain binds a nucleotide and subsequently transfers it to the active site (a conformational change known as “fingers closing”). For KF, it was shown that the fingers domain does not entirely close when non-complementary nucleotides are present, suggesting that nucleotides are screened for complementarity with the templating base during fingers closing. To see whether POLB employs a similar mechanism, I designed an smFRET assay with an immobile donor fluorophore on the DNA primer and an acceptor fluorophore on the fingers domain. Using this approach, I can visualize fingers closing in the presence of the correct nucleotide in single POLB-DNA complexes. Incorrect nucleotides (non-complementary dGTPs and complementary rUTPs) did not induce fingers closing. Instead, we observed a slight shift in the mean FRET efficiency of the open conformation (from E* ≈ 0.55 to E* ≈ 0.62), while a fully closed conformation corresponds to E* ≈ 0.75. I find evidence for a partially closed, fidelity-related conformation of the fingers subdomain. Simultaneously, I find that high concentrations of incorrect nucleotides (1 mM and 3 mM) stabilize the POLB-DNA complex by lowering the POLB dissociation rate. In contrast, for KF, a destabilizing effect was shown previously. The mechanism behind this stabilization remains unknown, but I hypothesize that with the abundance of incorrect nucleotides in the cell, DNA repair is much faster if high levels of these nucleotides do not promote dissociation.
In chapter 5, I introduce novel nanofluidic devices for high-throughput single-molecule imaging. These devices are completely made of glass. I present two designs: one design with a parallel array of nanochannels for equilibrium studies, and another with a single, T-shaped nanochannel for mixing studies allowing access to non-equilibrium conditions. A channel height of 200 nm confines movement of the molecules such that they do not move out of focus. With the implementation of parallel flow control, the devices can be driven with conventional syringe pumps. I achieve a high temporal resolution on our emCCD camera due to stroboscopic excitation (1.5 ms excitation in 10 ms frame time). I show that we can track single molecules at low concentrations for extended periods of time. The track length depends on the flow speed, but ranges from several frames to tens of frames. Moreover, at higher concentrations, I achieve hundreds of thousands of localizations within 10 minutes. These localizations allowed me to construct flow profiles, which confirms that the flow in the nanochannels is laminar. I also calculate that, at low flow rates and with the small DNA molecules I used, motion due to flow is of the same order of magnitude as motion due to diffusion. I illustrate this concept by mixing DNA hairpins in a primarily open configuration with a high-salt solution in the mixing channel: the FRET signature of the hairpins changes abruptly towards an equilibrium of primarily closed DNA hairpins. After fine-tuning the conditions, this so-called “diffusive” mixing is employed to trigger single-molecule reactions: I successfully polymerize my previously described DNA sensor inside the channel. I believe that these nanofluidic devices are a promising platform for studying non-immobilized single molecules at high throughput and high temporal resolution.
|Qualification||Doctor of Philosophy|
|Award date||21 Mar 2018|
|Place of Publication||Wageningen|
|Publication status||Published - 2018|
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