A single-molecule FRET sensor for monitoring DNA synthesis in real time

We developed a versatile DNA assay and framework for monitoring polymerization of DNA in real time and at the single-molecule level

Structural and kinetic characterization of DNA polymerases I and III from Escherichia coli

DNA elongation is performed by Pol III α subunit in E. coli, stimulated by the association with ε and θ subunits. These three subunits define the DNA Pol III catalytic core. There is controversy about the DNA Pol III assembly for the simultaneous control of lagging and leading strands replication, since some Authors propose a dimeric model with two cores, whereas others have assembled in vitro a trimeric DNA Pol III with a third catalytic core, which increases the efficiency of DNA replication. Moreover, the function of the PHP domain, located at the N-terminus of α subunit, is still unknown. Previous studies hypothesized a possible pyrophosphatase activity, not confirmed yet. The present Thesis highlights by the first time the production in vivo of a trimeric E. coli DNA Pol III by co-expressing α, τ, ε and θ subunits. This trimeric complex has been enzymatically characterized and a molecular model has been proposed, with 2 α subunits sustaining the lagging-strand replication whereas the third core replicates the leading strand. In addition, the pyrophosphatase activity of the PHP domain has been confirmed. This activity involves, at least, the H12 and the D19 residues, whereas the D201 regulates phosphate release. On the other hand, an artificial polymerase (HoLaMa), designed by deleting the exonuclease domain of Klenow Fragment, has been expressed, purified and characterized for a better understanding of bacterial polymerases mechanism. The absence of exonuclease domain impaired enzyme processivity, since this domain is involved in DNA binding. Finally, Klenow enzyme, HoLaMa, α subunit and DNA Pol III αεθ have been characterized at the single-molecule level by FRET analysis, combining ALEX and TIRF microscopy. Fluorescently-labeled DNA molecules were immobilized, and changes in FRET efficiency enabled us to study polymerase binding and DNA polymerization

HoLaMa: A Klenow sub-fragment lacking the 3′-5′ exonuclease domain

The design, construction, overexpression, and purification of a Klenow sub-fragment lacking the 30–50 exonuclease domain is presented here. In particular, a synthetic gene coding for the residues 515–928 of Escherichia coli DNA polymerase I was constructed. To improve the solubility and stability of the corresponding protein, the synthetic gene was designed to contain 11 site-specific substitutions. The gene was inserted into the pBADHis expression vector, generating 2 identical Klenow sub-fragments, bearing or not a hexahistidine tag. Both these Klenow sub-fragments, denominated HoLaMa and HoLaMaHis, were purified, and their catalytic properties were compared to those of Klenow enzyme. When DNA polymerase activity was assayed under processive conditions, the Klenow enzyme performed much better than HoLaMa and HoLaMaHis. However, when DNA polymerase activity was assayed under distributive conditions, the initial velocity of the reaction catalyzed by HoLaMa was comparable to that observed in the presence of Klenow enzyme. In particular, under distributive conditions HoLaMa was found to strongly prefer dsDNAs bearing a short template overhang, to the length of which the Klenow enzyme was relatively insensitive. Overall, our observations indicate that the exonuclease domain of the Klenow enzyme, besides its proofreading activity, does significantly contribute to the catalytic efficiency of DNA elongation