The thumb domain is not essential for the catalytic action of HoLaMa DNA polymerase

Abstract

A structural and kinetic characterization of a fragment of the HoLaMa DNA polymerase is presented here. In particular, a truncated form of HoLaMa, devoid of a consistent portion of the thumb domain, was isolated and purified. This HoLaMa fragment, denoted as ΔNter-HoLaMa, is surprisingly competent in catalyzing DNA extension, albeit featuring a k one order of magnitude lower than the corresponding kinetic constant of its full-length counterpart. The conformational rearrangements, if any, of enzyme tryptophanes triggered by DNA binding or extension were assayed under pre-steady-state conditions. The fluorescence of HoLaMa tryptophanes was found to significantly change upon DNA binding and extension. On the contrary, no fluorescence changes of ΔNter-HoLaMa tryptophanes were detected under the same conditions, suggesting that major conformational transitions are not required for DNA binding or extension by this truncated DNA polymerase.

Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain

“…This value is about two-fold higher compared to that previously determined for the binding of wild-type HoLaMa to DNA, i.e. k obs = (64 ± 10) s -1 [26]. However, it is important to remark that when the wild-type enzyme was assayed with the same DNA, we observed a fluorescence decrease [26], in agreement with the observations reported here performed in thermodynamic equilibrium (Fig 2B).…”

“…In addition, when the same ratio was higher than 1, the detected fluorescence was not further altered, suggesting that the enzyme was completely saturated with DNA (Fig 2B). This decrease in enzyme fluorescence triggered by the binding to 40mer polyA is in agreement with our previous kinetic assays, performed to detect the fluorescence changes of HoLaMa tryptophanes occurring after DNA binding [26]. Moreover, fitting the equation described by Sachs et al [33] (see Methods) to the experimental data, we estimated the number of DNA molecules bound to the enzyme, n , as n = 1.24 ± 0.03, and the dissociation constant, K D , of the enzyme-DNA complex as equal to K D = (35.1 ± 6.1) nM, in reasonable agreement with the 67 ± 19 nM value we previously reported [25].…”

“…k obs = (64 ± 10) s -1 [26]. However, it is important to remark that when the wild-type enzyme was assayed with the same DNA, we observed a fluorescence decrease [26], in agreement with the observations reported here performed in thermodynamic equilibrium (Fig 2B). Accordingly, the conformational rearrangements induced by DNA binding are mainly sensed by W866, and the fluorescence of this tryptophan may therefore be responsible for the signal used here to determine the stability of the HoLaMa-DNA complex (Fig 2C).…”

“…All experiments were performed using Escherichia coli TOP10 (genotype: F - mcrA Δ( mrr-hsdRMS-mcrBC ) ϕ80 lacZΔM15 Δ lacX74 recA1 araD139 Δ( ara-leu ) 7697 galU galK rpsL endA1 nupG ). For the overexpression of the HoLaMa enzyme, the procedure as previously described was used [26]: briefly, E . coli TOP10/pBAD-HoLaMa was grown for 9 h at 30°C in LB medium supplemented with 100 μg/mL ampicillin, and the overexpression of the target protein was induced for 15 h time interval, at the same temperature, with 1 mM arabinose.…”