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

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

“…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].…”

“…Next, we tested whether the association with DNA does affect or not the thermodynamic parameters diagnostic of HoLaMa stability. First, we excited at 280 nm and detected the emission at 328 nm of samples containing 500 nM HoLaMa and increasing concentrations of the 40mer dsDNA (40mer polyA, Fig 3), previously used to determine the extension activity exerted by HoLaMa [25]. In the presence of DNA/enzyme concentration ratios lower than 1, the fluorescence was found to decrease according to a hyperbolic behavior (Fig 2B).…”

“…Pioneering attempts to construct Klenow sub-fragments devoid of the 3’-5’ exonuclease domain revealed that the truncation of this domain conferred poor solubility to the majority of the variants designed [34,35]. Accordingly, the production of a properly folded Klenow sub-fragment lacking the proofreading domain requires a consistent re-engineering of the polymerase domain [25,35], and the successful preparation of Klenow sub-fragments whose DNA polymerase activity could be detected has been previously described [25,35]. However, no structural characterization was reported for any of these enzyme variants.…”

“…To address this point, we recently constructed a Klenow sub-fragment, i.e. a truncated form of Klenow devoid of the 3’-5’ exonuclease domain [25]. This sub-fragment, denoted HoLaMa, was shown to be competent in catalysing DNA extension, albeit with a lower efficiency when compared to its parental counterpart.…”

“…This sub-fragment, denoted HoLaMa, was shown to be competent in catalysing DNA extension, albeit with a lower efficiency when compared to its parental counterpart. In addition, we did also report that the binding of DNA substrates by HoLaMa occurs with similar affinities to those determined for Klenow enzyme [25]. However, it was not investigated neither the intrinsic overall thermodynamic stability of HoLaMa, nor the effects, if any, on enzyme stability triggered by the binding of DNA, so far.…”