A novel processive mechanism for DNA synthesis revealed by structure, modeling and mutagenesis of the accessory subunit of human mitochondrial DNA polymerase

Abstract

Mitochondrial DNA polymerase (pol gamma) is the sole DNA polymerase responsible for replication and repair of animal mitochondrial DNA. Here, we address the molecular mechanism by which the human holoenzyme achieves high processivity in nucleotide polymerization. We have determined the crystal structure of human pol gamma-beta, the accessory subunit that binds with high affinity to the catalytic core, pol gamma-alpha, to stimulate its activity and enhance holoenzyme processivity. We find that human pol gamma-beta shares a high level of structural similarity to class IIa aminoacyl tRNA synthetases, and forms a dimer in the crystal. A human pol gamma/DNA complex model was developed using the structures of the pol gamma-beta dimer and the bacteriophage T7 DNA polymerase ternary complex, which suggests multiple regions of subunit interaction between pol gamma-beta and the human catalytic core that allow it to encircle the newly synthesized double-stranded DNA, and thereby enhance DNA binding affinity and holoenzyme processivity. Biochemical properties of a novel set of human pol gamma-beta mutants are explained by and test the model, and elucidate the role of the accessory subunit as a novel type of processivity factor in stimulating pol gamma activity and in enhancing processivity.

Figure 1

Human pol γ-β shares structural homology with E. coli threonyl tRNA synthetase (ThrRS). (a) Ribbon display of the human pol γ-β structure. The N and C-terminal domains and HLH-β3 domain of monomer A are colored light green, green, and cyan, respectively. Monomer B is in gray. (b) Superposition of the N-terminal (upper panel) and C-terminal (lower panel) domains of human pol γ-β (blue) on the catalytic and anticodon binding domains of ThrRS, respectively. (c) RNA operator (yellow, PDB code 1KOG), tRNA (green, PDB code 1QF6), and DNA (orange, PDB code 1T7P), were docked onto the electrostatic surface of the pol γ-β dimer. (d) The electrostatic surface of the ThrRS dimer with bound tRNA (green) and RNA operator (yellow). Surfaces in (c) and (d) are colored according to electrostatic potential (red, −7.5kT/e⁻ to blue, +7.5kT/e⁻).

Figure 2

Structural model of the pol γ holoenzyme/ template-primer DNA complex. Bacteriophage T7 DNA polymerase in a ternary complex with template–primer DNA and ddGTP was docked onto the surface of the accessory subunit dimer of human pol γ in three steps as described in Materials and Methods. (a) Overview of the pol γ/DNA complex. (b) A partial view of the complex looking down the dsDNA helix. The dsDNA is represented by the magenta spiral and bases. Here, pol γ-β is presented in backbone structure with monomers colored green and yellow, and mutated residues (see the text) colored as follows: class I, black; class II, magenta; class IV, blue; and class V, red (class III not shown). T7 pol is in blue ribbon with the exonuclease domain in orange; P, F, T and E designate the palm, fingers and thumb subdomains, and the exonuclease domain, respectively. Thioredoxin in the T7 complex is shown in gray for comparison, and is not part of the pol γ holoenzyme.

Figure 3

Amino acid sequence alignment of pol γ-β. Amino acid sequence alignment of human (Hs), mouse (Mm) and Drosophila melanogaster (Dm) pol γ-β. Residues are shaded according to the degree of similarity, with dark gray shading indicating identical residues, medium gray shading indicating conservative substitutions and light gray shading indicating loosely conserved residues. Mutagenized residues in human pol γ-β are indicated in bold, and are colored according to class (see Figure 2 and Table 2), with correspondingly colored arrows indicating single mutants, and brackets indicating double mutants: class I, black; class II, magenta; class III, yellow (D433); class IV, blue; and class V, red.

Figure 4

Site-directed mutants of human pol γ-β show reduced stimulation of the pol γ-α catalytic core. (a) SDS-PAGE of wild-type and mutant forms of human pol γ-β; proteins were stained with silver. Fractions were assayed at five and tenfold molar excess over the catalytic core on singly-primed M13 DNA under standard conditions at 100 mM KCl, and the data from triplicate samples were averaged to determine the level of stimulation of DNA synthesis above that of the core alone. Roman numerals within brackets indicate the three structural domains of human pol γ-β. (b) Peak fractions obtained upon glycerol gradient sedimentation of human pol γ reconstituted with wild-type pol γ-α and the indicated mutant forms of pol γ-β that are representative of the low and moderate DNA binding affinity mutants in classes III and V (see Table 2).

Figure 5

Structural details of human pol γ-β mutants. (a) Mutant residues in the N-terminal domain of pol γ-β. (b) Local environment around residues E105 and G103 of pol γ-β. (c) Mutant residues in the C-terminal domain of pol γ-β. (d) Local charge balance at residue D433 in pol γ-β. Mutant residues are shown in color (see Figure 2 and Table 2): class I, black; class II, magenta; class III, yellow (D433); class IV, blue; and class V, red.

Figure 6

DNA binding affinity of human pol γ reconstituted with mutant forms of pol γ-β. Quantitative EMSA was performed using a P-labeled 21/45-mer primer-template and wild-type catalytic subunit (pol γ-α) reconstituted with various mutant forms of accessory subunit (pol γ-β) under standard conditions of DNA polymerase assay in the absence of dNTPs as described in Materials and Methods. Reactions were incubated for 1 min at 30 8C, and protein:DNA complexes were fractionated by electrophoresis in non-denaturing 6% polyacrylamide gels. Gels were dried and subjected to PhosphorImager analysis. (a) Representative EMSA analyses of strong (left), moderate (middle) and weak (right) DNA-binding mutant holoenzymes. Lanes indicated as pol γ-α contain the catalytic subunit alone, which results in a band shift that is distinct from that of pol γ holoenzyme shown in lanes indicated as pol γ. The remaining lanes represent titrations of reconstituted pol γ holoenzymes containing the indicated forms of mutant pol γ-β. (b) Protein titration data for 19 mutant holoenzymes.

Figure 7

Processivity of human pol γ reconstituted with mutant forms of pol γ-β. Human pol γ was reconstituted using a threefold molar excess of various mutant forms of the accessory subunit over wild-type pol γ-α, and DNA synthesis was measured at 100 mM KCl on singly primed M13 DNA. DNA product strands were isolated, denatured and electrophoresed in denaturing 1.5% agarose (upper panel) and 6% polyacrylamide (lower panel) gels, and the gels were exposed to a Phosphor Screen. Data (19 distinct bands) were quantified as described in Materials and Methods, and yielded the processivity values that are presented in Table 2.