Plant organellar DNA polymerases are replicative and translesion DNA synthesis polymerases

Abstract

Genomes acquire lesions that can block the replication fork and some lesions must be bypassed to allow survival. The nuclear genome of flowering plants encodes two family-A DNA polymerases (DNAPs), the result of a duplication event, that are the sole DNAPs in plant organelles. These DNAPs, dubbed Plant Organellar Polymerases (POPs), resemble the Klenow fragment of bacterial DNAP I and are not related to metazoan and fungal mitochondrial DNAPs. Herein we report that replicative POPs from the plant model Arabidopsis thaliana (AtPolI) efficiently bypass one the most insidious DNA lesions, an apurinic/apyrimidinic (AP) site. AtPolIs accomplish lesion bypass with high catalytic efficiency during nucleotide insertion and extension. Lesion bypass depends on two unique polymerization domain insertions evolutionarily unrelated to the insertions responsible for lesion bypass by DNAP θ, an analogous lesion bypass polymerase. AtPolIs exhibit an insertion fidelity that ranks between the fidelity of replicative and lesion bypass DNAPs, moderate 3'-5' exonuclease activity and strong strand-displacement. AtPolIs are the first known example of a family-A DNAP evolved to function in both DNA replication and lesion bypass. The lesion bypass capabilities of POPs may be required to prevent replication fork collapse in plant organelles.

Figure 1.

AtPolIs are family-A DNA polymerases (DNAPs) with canonical editing and polymerization domains. (A) Domain organization of AtPolIs in comparison to KF-DNAP I and Klen-Taq. AtPolIs contain a conserved 3′-5′ exonuclease and polymerization domains. The mitochondrial targeting sequence (MTS) of the dual targeting element is colored in gold and the predicted N-terminal disorder sequence is colored in cyan. (B) Homology model of AtPolIB in comparison to the crystal structure of Klen-Taq in complex with dsDNA and incoming nucleotide (upper part). Homology model of AtPolIB in comparison to the crystal structure of the KF-DNAP I with dsDNA and extruded 3′ single-stranded DNA in the exonuclease domain (bottom part). In both models the primer strand is colored in magenta and the template strand in gold. The incoming dNTP and the 3′ single-stranded DNA is black colored and in a ball-stick representation. The three unique amino acid insertions in POPs are colored in gray, blue and yellow.

Figure 2.

Phylogenetic analysis of POPs and heterologous purification of AtPolIs. (A) Phylogenetic tree constructed from POPs from land plants, algae and protozoan. Representative bacterial and T-odd bacteriophage DNAPs were included for comparison. A multiple sequence alignment was used to construct a phylogenetic tree using the neighbor joining algorithm, bootstrap values were calculated from 1000 trials and the evolutionary distances were constructed using the Poisson correction method. The bar indicates the numbers of substitutions per site. The significance of each branch of the phylogenetic tree is indicated by its bootstrap percentage. (B) 10% Coomassie blue stained SDS-PAGE gel showing the purification of full-length AtPolIA and AtPolIB after three purification steps: IMAC, phosphocellulose and heparin chromatography AtPolIs were purified as a single protein bands of ∼115 kDa.

Figure 3.

AtPolIs present 3′–5′ exonuclease and 5′–3′ polymerization activities. Exonuclease and polymerization activities measured using a 5′-³²P-labeled 24-mer primer annealed to a complementary 45-mer template DNA. (A) Equimolar amounts of each DNAP were incubated with the labeled primer-template and 3′–5′ exonucleolysis was initiated with the addition of MgCl₂ in the absence of dNTPs. Reactions were stopped at 15, 30, 60, 120 and 240 s. At first-time point (15 s) exonucleolytic degradation of the 24-mer to a 2-mer is observed in the samples incubated with T7DNAP (lane 11), whereas in the samples incubated with AtPolIA and AtPolIB degradation bands corresponding to a 17-mer and a 20-mer are present. The lane labeled with the minus sign (−) corresponds to the reaction without added MgCl₂. (B) Time course reaction from 15 to 240 s showing the polymerase activity of AtPolIA and AtPolIB in comparison to T7DNAP. 5′–3′ polymerization was initiated with the addition of 2 mM MgCl₂ and 100 μM of each dNTP. At the first-time point (15 s), full primer extension to a 45-mer is observed in the samples incubated with T7DNAP and dNTPs (lanes 11–14). In contrast the samples incubated with AtPolIA and AtPolIB present a constant accumulation of the 45-mer from 15 s to 4 min (lanes 1–10).

Figure 4.

AtPolIs perform strand-displacement DNA synthesis. (A) Time course nucleotide addition reaction from 15 to 240 s in a substrate assembled by hybridizing a 65-mer template to an extending 24-mer and blocking 35-mer (middle panel). This arrangement creates a gap of 6 nts before the blocking oligonucleotide. In reactions incubated with T7 DNAP a radioactively labeled band of 30 nts is predominantly observed, indicating that this polymerase is deficient in strand-displacement (lanes 11–15). In the reaction with AtPolIA, an accumulation of a product of 32 nts is observed (lanes 1–5) and only 9% of this product is able to reach the end of the template after an incubation of 240 s. In contrast, 32% of the product extended by AtPolIB is able to reach the end of the template after an incubation of 240 s (lanes 6–10). (B) Percentage of products synthetized by DNAPs after 240 s. The bands equal of lower to 24-mer correspond to substrate and exonucleolytic degradation, the bands from 25 to 30 nts correspond to gap filling and bands longer than 30 nts correspond to strand-displacement.

Figure 5.

AtPolIs bypass an apurinic/apyrimidinic (AP) site. (A) Time-course nucleotide incorporation reaction, from 15 s to 1 min, showing primer extension by AtPolIA, AtPolIB and AtPolIB exo⁻, in comparison to KF-DNAP I in canonical (lanes 1–9) or damaged templates (lanes 10–21) in the presence of 0.1 nM DNAP and 2 nM DNA. The template strand used for primer extension contained a non-damaged base or an AP site (tetrahydrofuran) 6 nts after the 3′-OH end of the primer. The migration of the 21-mer substrate and full-length extension 59-mer product are indicated in the gel. (B) Graphical representation of the lesion bypass efficiency by AtPolIs in comparison to KF-DNAP I. The values represent the relative amounts of diverse DNAs present in the reactions after 1 min. The products are subdivided into three classes: approach (before the AP site), insertion (incorporation opposite the AP site) and extension (nucleotide incorporation pass the AP site). In this representation the total amount of substrate and products is equal to 100%. Data represent the mean of three independent experiments.

Figure 6.

AtPolIs are low-fidelity DNAPs that bypass an AP site following the A rule. (A) Nucleotide insertion opposite a canonical template and an AP site. (A) Single nucleotide primer extension reactions using a template containing a canonical (dGMP) (lanes 1–5) or an AP site (lanes 6–10) following the 3′-OH end of the primer. In the reactions incubated with AtPolIB in the presence of dATP, dTTP and dGTP opposite a template G a band corresponding to nucleotide addition is synthetized (lanes 1–5). In the presence of dGTP, this band can be further extended (lane 5). In the reaction opposite a non-instructional AP site, dAMP is preferentially incorporated. dGMP and dTTP are incorporated with similar efficiencies, but dCTP is not efficiently used as a substrate (lanes 6–10). (B) AtPolIB is a low fidelity enzyme. Nucleotide incorporation by AtPolIB opposite all template bases. The identity of the template base is indicated by an X and the incorporated dNMP (A, T, G, C) is indicated. In all substrates, erroneous base incorporation and extension is observed. Incoming dNTPs were present at 100 μM.

Figure 7.

Specific amino acid insertions in AtPolI allow intrinsic lesion-bypass. Time course (from 15 s to 2 min) primer extension reaction by AtPolIB exo⁻, individual deletion mutants and KF-DNAP I using an undamaged or an AP site template. (A) primer extension on an undamaged template. (B) Primer extension on a template in which the AP site is the first nucleotide to be replicated. (C) Primer extension on a template in which the AP site is covered by 3′-AMP. Structural models of AtPolIB and each of the deletions mutants are depicted at the upper part of the figure. The amino acids corresponding to each of the three insertions are represented as spheres. The migration of the substrate and products are indicated in the gel. Reactions using a canonical template contained 0.1 nM DNAP and 2 nM DNA, whereas reactions in an AP site contained 1 nM DNAP. The migration of the primers located to hybridized before the lesion (29-mer) or to cover the AP site (30-mer) are indicated by asterisks.

Figure 8.

AtPolIB and POLQ present structurally equivalent insertions. Structural comparison between POLQ and AtPolIB. (A) Crystal structure of POLQ showing the interaction between R2254 and the 3′ phosphate of the primer strand. No continuous electron density was observed for regions corresponding to insertions 1 and 3. dsDNA is colored in orange and the palm, thumb and fingers subdomains are colored in red, green and cyan respectively. Structural model of AtPolIB showing the structural localization of its three insertions. Insertion 3 of AtPolIB is in a similar region that insertion 2 of POLQ. (B) Graphical representation of the insertions in POLQ and AtPolIB. In Both enzymes, insertion 1 is located at the thumb subdomain. POLQ insertions 2 and 3 are located at the palm subdomain, whereas in AtPolIB they are located at the thumb and fingers subdomain respectively.