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

Date Published: April 10, 2019

Publisher: Public Library of Science

Author(s): Michael Kovermann, Alessandra Stefan, Anna Castaldo, Sara Caramia, Alejandro Hochkoeppler, Giovanni Maga.


We report here on the stability and catalytic properties of the HoLaMa DNA polymerase, a Klenow sub-fragment lacking the 3’-5’ exonuclease domain. HoLaMa was overexpressed in Escherichia coli, and the enzyme was purified by means of standard chromatographic techniques. High-resolution NMR experiments revealed that HoLaMa is properly folded at pH 8.0 and 20°C. In addition, urea induced a cooperative folding to unfolding transition of HoLaMa, possessing an overall thermodynamic stability and a transition midpoint featuring ΔG and CM equal to (15.7 ± 1.9) kJ/mol and (3.5 ± 0.6) M, respectively. When the catalytic performances of HoLaMa were compared to those featured by the Klenow enzyme, we did observe a 10-fold lower catalytic efficiency by the HoLaMa enzyme. Surprisingly, HoLaMa and Klenow DNA polymerases possess markedly different sensitivities in competitive inhibition assays performed to test the effect of single dNTPs.

Partial Text

Despite large differences among their catalytic efficiencies, all DNA polymerases known so far share a peculiar molecular architecture, resembling an open right hand [1,2]. The domains of this molecular architecture are accordingly denoted as thumb, palm, and fingers, each one performing a particular function. The binding of a double-stranded DNA (dsDNA) substrate by the enzyme is mainly accomplished by the thumb domain [3], with the fingers domain subsequently binding a deoxynucleoside triphosphate (dNTP) [4] and two Mg2+ atoms. First, a Mg2+-dNTP complex is bound and paired to the template DNA strand by the fingers domain, and the binding of a second divalent magnesium triggers the so-called fingers closure, i.e. a consistent conformational change leading to DNA elongation [5,6]. The palm domain can indeed catalyse DNA elongation only when the enzyme is in the closed conformation, promoting the nucleophilic attack by the 3’-OH of the primer strand to the α-phosphate of the incoming dNTP [2]. Recently, the requirement of a third divalent cation for the action of DNA polymerases was revealed, suggesting that this third metal ion might promote product formation [7]. Besides this catalytic action, the maintenance of genomes stability demands for a stringent containment of DNA replication errors, and an ensemble of concerted actions contribute to this containment. In particular, it was shown that DNA polymerases catalyse DNA extension featuring exquisite precision, with an estimated frequency of the incorporation of erroneous bases into DNA equal to 10−4 [8,9]. Nevertheless, this extraordinary fidelity is not sufficient to guarantee genomes stability, and accessory functions assist DNA polymerases to accomplish this task. DNA elongation can therefore be accompanied by 3’-5’ exonuclease activity, which is responsible for the excision of erroneously incorporated dNTPS. Elegant assays in Escherichia coli reported that this 3’-5’ proofreading action lowers the frequency of mismatches down to about 10−7 [10]. Remarkably, this frequency can be further reduced to 10−10 with the aid of different post-replicative repair systems [11,12].

We recently reported on the construction of the HoLaMa DNA polymerase, a Klenow sub-fragment lacking the 3’-5’ exonuclease domain [25]. This enzyme was obtained by designing a synthetic gene coding for the polymerase domain of Klenow and containing eleven mutations, the majority of which were necessary to stabilize and to confer appropriate solubility to the artificial HoLaMa DNA polymerase. Here, we show that this engineered DNA polymerase features a substantial overall thermodynamic stability, comparable to that which has been previously reported by others for the Klenow enzyme possessing the 3’-5’ exonuclease domain. In addition, we did surprisingly observe that the deletion of the proofreading domain in HoLaMa alters the sensitivity of Klenow to the competitive inhibition exerted by dNTPS. HoLaMa and Klenow responded in markedly different ways when exposed to dNTPs unnecessary, or necessary at low concentrations, for the extension of a DNA primer strand. Overall, the thermodynamic stability and the peculiar catalytic features of HoLaMa suggest that this enzyme represents a promising tool for DNA manipulation.