DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

Abstract

DNA polymerases (DNAPs) responsible for genome replication are highly faithful enzymes that nonetheless cannot deal with damaged DNA. In contrast, translesion synthesis (TLS) DNAPs are suitable for replicating modified template bases, although resulting in very low-fidelity products. Here we report the biochemical characterization of the temperate bacteriophage Bam35 DNA polymerase (B35DNAP), which belongs to the protein-primed subgroup of family B DNAPs, along with phage Φ29 and other viral and mobile element polymerases. B35DNAP is a highly faithful DNAP that can couple strand displacement to processive DNA synthesis. These properties allow it to perform multiple displacement amplification of plasmid DNA with a very low error rate. Despite its fidelity and proofreading activity, B35DNAP was able to successfully perform abasic site TLS without template realignment and inserting preferably an A opposite the abasic site (A rule). Moreover, deletion of the TPR2 subdomain, required for processivity, impaired primer extension beyond the abasic site. Taken together, these findings suggest that B35DNAP may perform faithful and processive genome replication in vivo and, when required, TLS of abasic sites.

Keywords: Bam35; abasic sites; isothermal DNA amplification; protein-primed DNA polymerase; translesion synthesis.

Fig. 1.

Bam35 DNA polymerase synthetic and degradative activities. Shown is denaturing PAGE analysis of primer extension assays with a matched (A and C) or mismatched (B and D) 3′-terminus as depicted above the gels. The assays were carried out for 10 min at 37 °C in the presence of 10 nM of either WT (B35DNAP, A and B) or exonuclease-deficient (B35DNAPexo^—, C and D) DNAPs and the indicated concentration of dNTPs. Positions of the 15-mer substrate and 33-mer product bands are indicated on the right.

Fig. S1.

Schematic representation of Φ29 and Bam35 DNA polymerase sequences. Conserved domains and subdomains are represented as colored boxes, and their length is indicated. Identical residues are highlighted in red, and residues involved in DNA binding and catalysis are indicated with an asterisk.

Fig. S2.

B35DNAP nucleotide insertion fidelity. Shown is nucleotide insertion preference by the B35DNAPexo^— mutant using the ^3′-GTT template sequence context, in the presence of increasing amounts of each dNTP as indicated. Reactions were incubated for 10 min at 37 °C.

Fig. 2.

Bam35 DNA polymerase can couple strand displacement and processive polymerization during rolling circle DNA replication. (A) Replication of primed M13 DNA was carried out as described in Materials and Methods in the presence of 40 μM each of the four dNTPs and 2.25–91 nM of B35DNAP (lanes 1–9) or 50 nM of Φ29DNAP (lane 11). After incubation at 37 °C for 20 min, the length of the synthesized DNA was analyzed by alkaline 0.7% agarose gel electrophoresis alongside a λ DNA ladder (lane 10) and autoradiography. M13 ssDNA unit length is indicated as well. (B) Multiple-displacement amplification of plasmidic DNA by B35DNAP. The assay was performed as described in Materials and Methods, in the presence of 0–10 ng of pUC19 plasmid DNA as input and 50 nM of B35DNAP (lanes 2–9). Linearized pUC19 plasmid (100 ng) was loaded in lane 1, and linear DNA fragments obtained after digesting Φ29 DNA with HindIII, used as DNA length markers (lane 10), are indicated on the right.

Fig. 3.

Spectra of single base changes by B35DNAP. Bases are colored in red (A), blue (C), green (T), and orange (G). Base insertions, deletions, and substitutions are indicated above the sequence with inverted triangles (▼), Greek delta (Δ), and the mutated base, respectively, maintaining the same color code. Position 1 is the first transcribed nucleotide of the lacZ gene.

Fig. 4.

Abasic site TLS by B35DNAP. (A) Denaturing PAGE analysis of primer extension products by B35DNAP and Φ29DNAP on two different sequence contexts (lanes 1–6 vs. 7–12) and in the absence (^3′-GTT, lanes 1–3; ^3′-GTA, lanes 7–9) or presence (^3′-GFT, lanes 4–6; 3′-GFA, lanes 10–12) of a THF (F) abasic site analog. The reactions, containing 1 nM of labeled duplex substrate, 10 nM of each enzyme, and either 100 nM or 100 μM of each dNTP for templates without or with an abasic site, respectively, were incubated for 30 min at 37 °C (Materials and Methods). Schematic representations of each template/primer substrate are depicted above. Positions of the 15-mer substrate and 16- and 33-mer products are indicated on the right. (B) Processive replication of primer/template substrates by B35DNAP. Assays were performed with excess (900 nM) of primer/template ^3′-GTT (lanes 1–9) or ^3′-GFT (lanes 10–18) substrates and decreasing concentrations of B35DNAP. Reactions were incubated for 30 min at 37 °C and loaded into denaturing PAGE. Positions of the 15-mer substrate and 16- and 33-mer products are indicated on the right.

Fig. S3.

Abasic site TLS by B35DNAP follows the A rule. Shown is nucleotide insertion preference opposite the THF by the B35DNAPexo^— mutant using the ^3′-GFT (A) or ^3′-GFA (B) template sequence context, in the presence of increasing amounts of each dNTP as indicated. Reactions were incubated for 10 min at 37 °C.

Fig. 5.

Time course analysis of abasic site TLS by B35DNAP. Assays were made with an excess of substrate (see text for details) and two alternative sequence contexts, ^3′-CTT and ^3′-CFT (A) or ^3′-CTA and ^3′-CFA (B), and with a 90:1 substrate:enzyme ratio. Under these conditions, maximum template utilization (at 30 min) was between 30--40%. Shown are mean and SE of three independent experiments of full-length replicated product in the absence of damage (gray line), or insertion probability (blue line) and bypass probability (red line), as well as relative full-length replication of the THF-containing template (black line).

Fig. 6.

Exonuclease activity counteracts primer extension beyond the THF. (A) Detailed template strand sequence as well as the alternative primers used are depicted above, where “X” stands for either T or THF nucleotide and the sequence context ^3′-GXT is highlighted. Polymerization activity by B35DNAP on a primer/template substrate without (lanes 1–10) or with THF (F) in the first template position (lanes 11–20) by sequential addition of 100 nM (lanes 1–10) or 100 μM (lanes 11–20) of the indicated dNTPs. (B) Similar primer extension assay by the B35DNAPexo^— in the presence of 50 nM (lanes 1–13) or 1 μM (lanes 14–26) of the indicated dNTPs. The effects of dNTP concentration on polymerization on undamaged and damaged templates by WT and exonuclease-deficient B35DNAP are shown in Fig. 1 and Fig. S4, respectively. Positions of the 15-mer substrate and the 19-, 22-, and 33-mer products are indicated.

Fig. 7.

Short-term memory of abasic sites containing mismatch by B35DNAP. (A) Detailed template strand sequence as well as the alternative primers used are depicted above the gel, where “X” represents either T or THF nucleotide. The relative distance of the X with respect to the primer size is also indicated on the right. Reactions were performed for 10 min at 37 °C in the presence of 10 nM B35DNAP and 100 μM dATP (lanes 9, 11, 16, and 18), dATP and dCTP (lanes 8, 12, 14, 15, 19, and 21), dATP and dGTP (lanes 10 and 17), or dCTP and dGTP (lanes 13 and 20). (B) B35DNAP replication of the same primer/template substrates but with 10 µM of the four dNTPs. Primer length is specified above the gel and positions of the 15- and 22-mer substrates and the 16- and 33-mer products are indicated on the right.

Fig. S4.

Effect of dNTP concentration on B35DNAP abasic site TLS. Primer extension assays of the ^3′-GFT sequence context are shown. The assays were carried out for 10 min at 37 °C in the presence of 10 nM of either WT (A) or exonuclease mutant (B) B35DNAP and the indicated concentration of all dNTPs. Positions of the 15-mer substrate and 33-mer product bands are indicated on the right.

Fig. S5.

Exonuclease and polymerase activities of B35DNAP ΔTPR2 mutant. Shown is the polymerization activity of the B35DNAP ΔTPR2 mutant (200 nM) on primer/template substrate in the absence (lane 5) or presence (lanes 6–10) of increasing concentrations of dNTPs. WT B35DNAP (10 nM) in the absence (lane 2) or presence (lane 3) of 100 nM dNTPs served as a control. Positions of the 15-mer substrate and 33-mer product bands are indicated on the right.

Fig 8.

The B35DNAP TPR2 motif is required for extension beyond the abasic site. Polymerization activity of WT (10 nM) and ΔTPR2 (200 nM) B35DNAP on primer/template substrates with or without a THF lesion, as depicted above. Reactions were performed for the indicated times at 37 °C in the presence of 100 μM dNTPs. Positions of the 15-mer substrate and the 16- and 33-mer products are indicated on the right.