ASFV DNA polymerase extends recessed DNAs with catalytic efficiencies outperforming those exerted on gapped DNA substrates

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Highlights

  • We compared the action of ASFV DNA polymerase on recessed or gapped DNAs.

  • Recessed DNAs are preferred to gapped substrates with the same extendable length.

  • Recessed DNAs bearing a short overhang are poor substrates.

  • DNA binding is rate limiting for both primer extension and gap filling activities.

Abstract

The DNA polymerase from african swine fever virus (ASFV Pol X), lacking both 8 kDa and thumb domains, is the smallest enzyme featuring competence in DNA extension. Here we show that ASFV Pol X features poor filling activity of DNA gaps consisting of 15 bases, and exerts a more efficient action at the expense of DNA substrates containing a recessed end of equal length. We also show that shortening the recessed end of DNA substrates decreases the rate of DNA elongation catalysed by ASFV Pol X. Finally, by means of stopped-flow experiments we were able to determine that DNA binding is a step responsible for restraining the efficiency of ASFV Pol X catalytic action.

Introduction

DNA polymerases catalyse the extension of DNA substrates accomplishing two main different tasks, the replication of genomes and the repair of damaged DNA [1]. These two functions are exerted by specialized enzymes, exclusively committed to genome replication or to repair sites containing impaired DNA. In addition, DNA polymerases can possess additional catalytic features, e.g. the 3′-5′ exonuclease activity [2], residing in the same polypeptide bearing polymerase activity [3] or in different subunits of a multimeric holoenzyme [4]. Independently of the diversity of actions exerted, all DNA polymerases share a peculiar molecular architecture resembling the shape of an open right hand [5,6]. Further, this molecular architecture is composed of three main protein domains (denoted thumb, palm, and fingers), which can be associated to specific functions necessary for DNA extension [[6], [7], [8]].

A notable exception to the highly conserved molecular architecture of DNA polymerases is represented by the enzyme from the African Swine Fever Virus (ASFV). This DNA polymerase does indeed lack the thumb domain [9,10] (Fig. 1A), and features a primary structure composed of 174 amino acids, accounting for a molecular mass equal to 20.3 kDa. Accordingly, the ASFV enzyme is the smallest enzyme among the DNA polymerases characterized so far.

Structurally speaking, the ASFV DNA polymerase (ASFV Pol) belongs to family X of DNA polymerases [11]. This protein family is composed of enzymes essential for DNA repair, i.e. DNA polymerases β, λ, and μ. Among these enzymes, ASFV DNA Pol features the highest structural similarity to DNA Pol β (Fig. 1B), which is competent in base excision repair (BER) and is provided with an additional 8 kDa domain contiguous to fingers domain [12]. The BER pathway does usually consist of 4 consecutive steps: i) the damaged base present at a DNA site to be repaired is removed by a DNA glycosylase; ii) the abasic site accordingly generated is incised by an abasic endonuclease; iii) 5′-deoxyribose is released from the abasic and incised site by the DNA Pol β lyase activity, exerted by the 8 kDa domain; iv) and finally, the gapped DNA is filled by DNA Pol β. Curiously enough, ASFV DNA Pol not only lacks the thumb domain, but is also devoid of the 8 kDa domain present in DNA Pol β [9,10]. However, it is interesting to note that ASFV DNA Pol was shown to be competent in performing endonucleolytic activity on unincised abasic sites [13]. In addition, it was demonstrated that the ASFV genome codes for an abasic endonuclease (protein pE296R) [14] and for a DNA ligase [15]. Overall, these observations indicate the occurrence of a peculiar BER mechanism responsible for the repair of damaged ASFV DNA. Further, this peculiarity does also apply to the activity of ASFV DNA Pol on gapped DNA substrates. It was indeed shown that the presence of a 5′-Pi in DNA gaps strongly enhances the enzyme gap filling activity [16]. Moreover, it was recently demonstrated that the association of ASFV DNA Pol to gapped substrates containing a 5′-Pi implies a set of interactions additional to those occurring with gapped DNAs devoid of a 5′-Pi [17]. In particular, 5 hydrogen bonds are formed between the enzyme and the 5′-Pi of the gapped substrate, supplementing the 11 hydrogen bonds between the DNA phosphate groups around the gap and the C81–K85 and Y135–Y140 enzyme regions [17].

Functionally speaking, ASFV DNA Pol was shown to exert activity on both recessed and gapped DNA substrates. In particular, it was demonstrated that: i) the enzyme features lower Km values for gapped DNAs, independently of the presence of a 5′-Pi [13]; ii) the kcat values determined in the presence of recessed DNAs are higher than those for gapped DNAs, unless a 5′-Pi is present in the substrate gap [13]. In addition, it was shown that ASFV DNA Pol prefers the incorporation of purines over pyrimidines [13,17], with KD values for purines one order of magnitude lower than those determined for pyrimidines [17]. Nevertheless, ASFV DNA Pol usually features a modest activity with kcat values ranging from 0.52•10−4 to 0.57•10−2 s−1 [13,18].

The catalytic efficiency of ASFV DNA Pol at the expense of recessed DNAs was compared quite a number of times with that determined in the presence of gapped DNA substrates. However, these comparisons were performed using heterogeneous substrates, i.e. recessed DNAs containing overhangs of 6–20 bases and DNAs containing a single-nucleotide gap [13,16,19]. Therefore, we thought it of interest to determine how the length of an overhang affects ASFV DNA Pol activity, and to compare the enzyme activity using DNA substrates containing overhangs or gaps of equal length and base composition. Here we report on these comparisons, along with an evaluation of the conformational rearrangements associated with binding of the enzyme to recessed or gapped DNAs.

Section snippets

Bacterial strain, plasmid and DNAs

Escherichia coli BL21 (DE3) was used for protein expression. The gene coding for ASFV Pol X, synthesized and optimized for E. coli codon usage by GenScript (Piscataway, NJ, USA), was cloned into pET19b plasmid using NcoI and BamHI restriction sites, yielding the pET19b-ASFVPolX construct. For the expression of ASFV Pol X bearing a decahistidine tag at the N-terminus, the synthetic gene was cloned into the same vector using NdeI and BamHI sites, yielding the pET19b-ASFVPolXhis construct. The

Results and discussion

First, we tested the overexpression of His-tagged and tagless ASFV Pol X by performing SDS-PAGE analyses of protein extracts isolated from cells induced or not with 1 mM IPTG. Both enzyme forms were overexpressed at high levels, and were mainly recovered from the soluble fraction of protein extracts isolated from induced cells (Fig. S2). Moreover, both His-tagged and tagless ASFV Pol X were conveniently purified to homogeneity (Fig. 1C), using procedures consisting of 2 and 3 purification

Concluding remarks

We show here that ASFV DNA polymerase X features poor activity when filling gaps of consistent length, i.e. 15 bases, performing much better when engaged in the extension of recessed ends of equal length (Table 1). According to our observations, both the extension of recessed ends and the gap-filling activity are rate limited by DNA binding. Therefore, we propose that the absence of both thumb and 8 kDa domains in ASFV Pol X restrains the DNA binding efficiency and the catalytic performance of

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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