Activity and fidelity of human DNA polymerase α depend on primer structure

Abstract

DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA-DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure-function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA-DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the "fingers" subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.

Keywords: DNA polymerase; DNA replication; DNA-protein interaction; Polα; Polδ; Polε; RNA; chimeric RNA-DNA primer; conformational change; crystal structure; human; pre-steady-state kinetics.

Figure 1.

Ternary complexes of p180core containing dCTP and DNA:DNA or DNA:RNA duplexes. A, overall view of the p180core/DNA:RNA/dCTP complex. The carbons of dCTP, DNA template, and RNA primer are colored yellow, marine, and purple, respectively. B, alignment of p180core ternary complexes containing RNA or DNA primers (PDB codes 4QCL and 6AS7, respectively). p180core, template:primer, and dCTP are colored gray in the complex containing DNA:DNA. In the bottom panel, DNA:DNA from the binary complex (PDB code 5IUD), which is superimposed onto p180core/DNA:RNA/dCTP with RMSD of 1.08 Å for 762 Cα atoms, is colored orange (for clarity, double helices are shown separately from p180core).

Figure 2.

Conformations of the fingers in hPolα. A, the open-fingers conformation in the aligned ternary hPolα/DNA:DNA/dCTP and binary hPolα/DNA:DNA complexes (PDB codes 6AS7 and 5IUD, respectively). B, the open-fingers conformation in the aligned complexes hPolα/DNA:DNA/dCTP and hPolα/DNA:RNA/aphidicolin (PDB codes 6AS7 and 5Q4V, respectively). C, switching of Ile⁹⁴⁴ between two sites upon fingers moving. In the aligned ternary complexes, the subdomains of hPolα in the complex with DNA:RNA and DNA:DNA (PDB codes 4QCL and 6AS7, respectively) are colored according to Fig. 1 and gray, respectively. Ile⁹⁴⁴ and the residues interacting with it (in different finger conformations) are shown as sticks. Leu⁷⁶⁴ interacts with Ile⁹⁴⁴ in either conformation and is shown on both aligned molecules. The arrow depicts the distance between Cα atoms of Leu⁹³⁴, located in the loop connecting two helices of the fingers. The substrates and aphidicolin are not shown for clarity.

Figure 3.

Analysis of two disordered regions in p180core. Ternary complexes of yPolδ and yPolϵ (PDB codes 3IAY and 4M8O, respectively) were superimposed onto p180core/DNA:RNA/dCTP with RMSD of 2.08 and 5.05 Å for 524 and 483 Cα atoms, respectively. Structured p180core regions are colored according to Fig. 1; unstructured regions are depicted by dashed gray lines. The yPolϵ regions 537–557 and 677–764 form the P-domain.

Figure 4.

Interaction of Polα with incoming dNTP. A, hydrophilic interactions between hPolα and the triphosphate moiety of dNTP. In aligned ternary complexes containing DNA:RNA and DNA:DNA duplexes (PDB codes 4QCL and 6AS7, respectively), there is a good superposition of dCTP-interacting residues except the fingers. The subdomains of hPolα in the complex with DNA:RNA are colored according to Fig. 1. Polα in the complex with DNA:DNA is colored gray (only α-helices of fingers are shown for clarity); dCTP from this complex is shown as lines and colored dark brown. In both complexes, the side or main chains of the hPolα residues involved in hydrophilic interactions with dCTP and metals are shown as sticks. Potential H-bonds between Polα (in complex with DNA:RNA), dCTP, and catalytic metals are depicted by pink dashed lines. The A and B metal-binding sites of hPolα in complex with DNA:RNA or DNA:DNA contain Zn²⁺ and Mg²⁺ or only Mg²⁺, respectively. Asp⁸⁶⁰ and Asp¹⁰⁰⁴ each interact with both divalent metals; the metal-B interacts with all phosphates of dNTP, whereas the metal-A makes a contact only with the α-phosphate. B, comparison of dNTP-binding pockets in hPolα and yPolδ. The ternary complexes hPolα/DNA:RNA/dCTP and yPolδ/DNA:DNA/dCTP (PDB codes 4QCL and 3IAY, respectively) were aligned with RMSD of 2.08 Å for 524 Cα atoms. The carbons of hPolα and yPolδ are colored according to Fig. 1 and gray, respectively. hPolα Phe⁸⁶¹ and yPolδ Phe⁶⁰⁹, as well as two water molecules coordinated by zinc are not shown for clarity. dCTP from Polδ complex is shown as lines and colored dark brown. Atoms of magnesium, calcium, and zinc are presented as spheres at reduced scale and colored green, light orange, and slate, respectively. A different conformation of Asp⁶⁰⁸ in yPolδ is probably due to the coordination of a third calcium ion. Accordingly, Asp⁸⁶⁰ of hPolα is oriented similarly to Asp⁶⁴⁰ in yPolϵ where Mg²⁺ occupies the site B (22). In both panels, DNA:DNA and DNA:RNA duplexes are not shown for clarity.

Figure 5.

The effect of finger mutations on hPolα activity depends on the divalent metal. Extension of dA₁₅ primer annealed to dT₇₀ was carried out in the presence of 30 nm p180core, 1.5 μm template:primer, 0.1 mm dATP, and specified divalent metal ions. The dashed line indicates splicing of the original image.

Figure 6.

Schematic diagrams of the interactions of eukaryotic replicative Pols with the sugar-phosphate backbone of a template:primer. The crystal structures of the following complexes were analyzed: hPolα/DNA:DNA/dCTP (A), hPolα/DNA:RNA/dCTP (B), hPolα/DNA:DNA (C), yPolα/DNA:RNA/dGTP (D), yPolδ/DNA:DNA/dCTP (E), and yPolϵ/DNA:DNA/dATP (F). Among residues making van der Waals interactions with a template:primer, only the arginines contacting the P₄ sugar are shown for clarity. Residues highlighted in italic type can make a hydrogen bond with a template:primer in their alternative conformation. Ribose, deoxyribose, and dideoxyribose rings are colored red, gray, and olive, respectively. The residues interacting with a template:primer are color-coded according to the subdomains they belong to (see Fig. 1 for color-coding). The conservative amino acids are underlined. The residues marked by an asterisk use a main-chain atom to establish H-bond with a sugar-phosphate backbone. The pound sign indicates that the residue makes double contact with a template:primer, using both the side- and main-chain atoms. The type of the double helix (A, B, or intermediate AB) was determined for each dinucleotide step using Web 3DNA software (Rutgers University) (69). This software requires the presence of two consecutive dinucleotide steps of the same type to confidently determine the helix type, so the third dinucleotide step of DNA:RNA was downgraded by the program to unclassified. We assume that in locally distorted double helices, the presence of a single isolated step may take place, which is in line with data on yPolα (16). In the p180core/DNA:RNA/aphidicolin complex (PDB code 4Q5V), the pattern of interactions between hPolα and the template:primer is the same as shown in B except for the loss of the H-bond between T₋₁ and Ser⁷⁸⁵.

Figure 7.

RNA primer bending. A, primer conformation in the complexes of hPolα with DNA:RNA and DNA:DNA. The p180core/DNA:RNA/dCTP and p180core/DNA:DNA complexes (PDB codes 4QCL and 5IUD, respectively) were aligned with RMSD of 0.5 Å using the palm and thumb. In the complex containing DNA:RNA, RNA primer and Arg¹⁰⁸² are colored according to Fig. 1. In the complex containing DNA:DNA, the carbons of a DNA primer and amino acids are colored gray and green, respectively. In the complexes containing RNA or DNA primer, amino acids are represented as sticks or lines, respectively. The modeled ribose with the 3′-endo pucker was superimposed on the P₄ sugar of the DNA primer and has the same position of carbons (colored yellow) except C2. The arrow depicts the difference in position of P₄ 2′-OH in the RNA primer and the modeled ribose. Pink dashed lines and double arrows depict H-bonds and distances between atoms, respectively. B, overall view of RNA primer in the complex p180core/DNA:RNA/dCTP.

Figure 8.

Conformation of the P₁ phosphate in the DNA and chimeric RNA–DNA primers. A, conformation of P₁-P₂ in the complexes of hPolα with DNA and chimeric primers. The p180core/DNA:RNA/dCTP and p180core/DNA:DNA complexes (PDB codes 4QCL and 5IUD, respectively) were aligned with RMSD of 0.42 Å using the palm. In the former complex, the RNA primer may be considered chimeric due to the presence of dideoxycytidine at the 3′-end. The carbons are colored gray in the complex of p180core with DNA:DNA. B, conformation of P₁-P₂ in the complexes of yPolδ and yPolα with DNA and chimeric primers, respectively. The ternary complexes of yPolα and yPolδ (PDB codes 4FYD and 3IAY, respectively) were aligned with RMSD of 0.67 Å using the palm. The carbons are colored gray in the complex of yPolδ with DNA:DNA. Blue dashed lines depict the H-bonds.

Figure 9.

Single-incorporation kinetics of hPolα. A, sequence of template:primers used for transient kinetic assays. B and C, burst kinetics assay on DNA and RNA primers, respectively. p180core (3 μm) was incubated with radiolabeled DNA (B) or RNA (C) primer (9 μm) annealed to a DNA template and rapidly mixed with dATP (200 μm). Product formation was plotted versus time, and a linear equation was fit to the data (red dashed line). D, single-nucleotide incorporation assay. p180core (3 μm) was incubated with radiolabeled DNA or RNA primer (100 nm) annealed to a DNA template and rapidly mixed with various concentrations of dATP (1–300 μm). Product formation was plotted versus time and fit to a single-exponential equation to obtain the observed rates of product formation. The rates were plotted against concentration of dATP and fit to a hyperbolic equation for both DNA primer (blue) and RNA primer (red) elongation to obtain K_d and k_pol values.

Figure 10.

Sequential incorporation modeling of hPolα kinetics. A, a representative gel of the elongation of the DNA primer substrate in the presence of 100 μm dATP and 2 μm dTTP. The n band represents the 15-mer primer; n + 1, the primer elongated by dATP across template dT; n + 2, the primer elongated first by dATP followed by dTTP across template dA. p180core (3 μm) was incubated with radiolabeled DNA template:primer (100 nm) and rapidly mixed with saturating concentrations of dATP (100 μm) and various concentrations of dTTP (1–100 μm). B, the time course of DNA primer elongation. p180core (3 μm) was incubated with radiolabeled DNA template:primer (100 nm) and rapidly mixed with saturating concentrations of dNTPs (240 μm). C, time course of RNA primer elongation. D, processivity modeling for DNA primer elongation. Product formation was plotted versus time, and the incorporations were fit to a processive kinetic model using KinTek Global Explorer to provide estimates of the k_pol and k_off values. The first five incorporations are shown. The red line represents the unelongated primer (15-mer). The inset shows a zoomed in view of the third through fifth incorporations. E, processivity modeling for RNA primer elongation. The dashed line shown in the inset represents the model if the rate of the fourth incorporation of the RNA assay was simulated to be identical to the rate of the fourth incorporation from the DNA processivity assay.

Figure 11.

Comparison of Polα fidelity in extension of DNA and RNA primers. A, template and primer sequences. DNA (B) and RNA (C) primers were extended with cognate and noncognate dNTPs. Reactions were carried out at 35 °C for 20 min and contained 20 nm p180core (except lane 1, which is a control reaction without enzyme), 1 μm template:primer, 2 mm MgCl₂, and 0.2 mm dNTP.

Figure 12.

Polα displays misincorporation on a rapid time scale. p180core (3 μm) was incubated with radiolabeled DNA template:primer substrate (10 μm) and rapidly mixed with saturating dATP (500 μm). The gel shows the elongation of the DNA primer in the presence of only dATP. The n + 1 band corresponds to a correct incorporation of dATP across from the templating dT. The n + 2 band corresponds to a misincorporation of dATP across from a templating dA. The sequence shows where the incoming dATP is incorporated onto the substrate.

Figure 13.

Alignment of hPolα/DNA:RNA/dCTP and yPolδ/DNA:DNA/dCTP. The ternary complexes were aligned as described in the legend to Fig. 4B. hPolα and DNA:RNA are colored according to Fig. 1. yPolδ and DNA:DNA are colored pale yellow and gray, respectively; the region spanning residues 895–900 of yPolδ is highlighted in blue. Two potential clash areas between Polδ and the RNA primer are depicted by red stars. The distant part of the 5′-overhang of DNA template complexed with yPolδ is not shown for clarity.

Figure 14.

Analysis of structural factors affecting Polα selectivity for dNTP. A, DNA template has significant conformational flexibility near T₀ in the complex with hPolα. Aphidicolin binding by Polα results in rotation of the T₀ base and the T₋₁ sugar by 112 and 168°, respectively. The p180core/DNA:RNA/dCTP and p180core/DNA:RNA/aphidicolin complexes (PDB codes 4QCL and 5Q4V, respectively) were aligned with RMSD of 0.27 Å using the palm. B, modeling of the A:dATP mispair in the hPolα active site. DNA carbons are colored slate and gray in the case of the ternary complexes with dCTP and aphidicolin, respectively. The carbons of modeled adenines at T₀ and in place of dCTP are colored green and cyan, respectively. C, the modeled 2′-OH has steric hindrance with Thr¹⁰⁰³. The coordinates of the p180core/DNA:RNA/dCTP complex (PDB code 4QCL) were used for modeling (the sugar of the 3′-dideoxycytidine has a 3′-endo pucker).