The accessory subunit of mtDNA polymerase shares structural homology with aminoacyl-tRNA synthetases: implications for a dual role as a primer recognition factor and processivity clamp

Abstract

The accessory subunit of the heterodimeric mtDNA polymerase (polgamma) from Drosophila embryos is required to maintain the structural integrity or catalytic efficiency of the holoenzyme. cDNAs for the accessory subunit from Drosophila, man, mouse, and rat have been identified, and comparative sequence alignment reveals that the C-terminal region of about 120 aa is the most conserved. Furthermore, we demonstrate that the accessory subunit of animal polgamma has both sequence and structural similarity with class IIa aminoacyl-tRNA synthetases. Based on sequence similarity and fold recognition followed by homology modeling, we have developed a model of the three-dimensional structure of the C-terminal region of the accessory subunit of polgamma. The model reveals a rare five-stranded beta-sheet surrounded by four alpha-helices with structural homology to the anticodon-binding domain of class IIa aminoacyl-tRNA synthetases. We postulate that the accessory subunit plays a role in the recognition of RNA primers in mtDNA replication, to recruit polgamma to the template-primer junction. A similar role is served by the gamma-complex in Escherichia coli DNA polymerase III, and indeed our accessory subunit model shows structural similarity with the N-terminal domain of the delta' subunit of the gamma-complex. Structural similarity is also found with E. coli thioredoxin, the accessory subunit and processivity factor in bacteriophage T7 DNA polymerase. Thus, we propose that the accessory subunit of polgamma is involved both in primer recognition and in processive DNA strand elongation.

Figure 1

Sequence comparisons of the accessory subunit of Drosophila polγ with mammalian homologs and with class IIa aaRSs. (A) Sequence alignment between the C-terminal regions of the accessory subunit of D. melanogaster polγ (Dm) (7) and its homologs from Homo sapiens (Hs) (7) and Mus musculus (Mm, GenBank accession no. AF006072). Residues are shaded based on degree of similarity, with dark shading indicating identical residues and light shading indicating conservative substitutions within the categories of the simplification matrix from the UW-GCG package (12): negatively-charged residues and derivatives (Asp, Asn, Glu, Gln); positively charged residues (His, Lys, Arg); small hydrophobic residues (Leu, Ile, Val, Met); large hydrophobic residues (Phe, Tyr, Trp); Cys; and other residues (Pro, Ala, Gly, Ser, Thr). Open boxes indicate loosely conserved residues, i.e., those conserved as hydrophobic or hydrophilic. ⋄ marks every 10th residue in the Drosophila polγ-β sequence. (B) Comparison of the Drosophila polγ-β sequence with the T. thermophilus (Tt) prolyl-RS (13), glycyl-RS (PDB ID 1ati) (24), and histidyl-RS sequences (PDB ID 1adj) (18). Sequences are shaded as in A, with underlined residues in polγ indicating conservation of hydrophobic/hydrophilic character between the accessory subunit and the RSs.

Figure 2

Structural modeling and comparison of the C-terminal domain of the accessory subunit of Drosophila polγ with other DNA polymerase accessory subunits. (A) Molecular model of the C-terminal domain of Drosophila polγ-β. The main-chain ribbon is shown along with protein side-chain atoms colored according to chemistry: negatively charged residues and derivatives (Asp, Asn, Glu, Gln), red; positively charged residues (His, Lys, Arg), blue; small hydrophobic residues (Leu, Ile, Val, Met), green; large hydrophobic residues (Phe, Tyr, Trp), purple; cysteine, yellow; and other residues (Pro, Ala, Gly, Ser, Thr), gray. The termini of the α-helices and β-strands are numbered according to the Drosophila polγ-β sequence in Fig. 1, with the N and C termini labeled as N and C. (B) Comparison of the structures of the modeled C-terminal domain of Drosophila polγ-β and the C-terminal domain of prolyl-RS, used as a template for the polγ-β model. The main-chain structure of the model, oriented as in A, is shown superimposed onto the anticodon-binding domain of T. thermophilus prolyl-RS. The accessory subunit is colored according to sequence conservation between the Drosophila and human sequences; residues conserved between the two sequences are shown in yellow along with their surface-accessible side chains, conservatively substituted residues are in green, nonconserved residues are in blue, and insertions in the fly sequence relative to the human sequence are in red. (There are no deletions in the fly sequence relative to human.) Conservation is defined as in Fig. 1. The prolyl-RS main-chain ribbon is colored by sequence conservation among the three related aaRSs shown in Fig. 1, with conserved residues in dark pink, similar residues in light pink, and nonconserved residues in grey. (C) Comparison of the structures of the accessory subunit of polγ, the δ′-subunit of E. coli DNA polymerase III, and E. coli thioredoxin. The N-terminal domain of DNA polymerase III δ′ (PDB ID 1a5t; ref. 36) is shown in blue, polγ-β in magenta, and thioredoxin (PDB ID 1t7p, chain B) (40) in green, with thioredoxin residues interacting with the catalytic subunit of T7 DNA polymerase shown in yellow. Optimally similar orientations were produced by using dali (22, 47).