The accessory subunit of Xenopus laevis mitochondrial DNA polymerase gamma increases processivity of the catalytic subunit of human DNA polymerase gamma and is related to class II aminoacyl-tRNA synthetases

Abstract

Peptide sequences obtained from the accessory subunit of Xenopus laevis mitochondrial DNA (mtDNA) polymerase gamma (pol gamma) were used to clone the cDNA encoding this protein. Amino-terminal sequencing of the mitochondrial protein indicated the presence of a 44-amino-acid mitochondrial targeting sequence, leaving a predicted mature protein with 419 amino acids and a molecular mass of 47.3 kDa. This protein is associated with the larger, catalytic subunit in preparations of active mtDNA polymerase. The small subunit exhibits homology to its human, mouse, and Drosophila counterparts. Interestingly, significant homology to glycyl-tRNA synthetases from prokaryotic organisms reveals a likely evolutionary relationship. Since attempts to produce an enzymatically active recombinant catalytic subunit of Xenopus DNA pol gamma have not been successful, we tested the effects of adding the small subunit of the Xenopus enzyme to the catalytic subunit of human DNA pol gamma purified from baculovirus-infected insect cells. These experiments provide the first functional evidence that the small subunit of DNA pol gamma stimulates processive DNA synthesis by the human catalytic subunit under physiological salt conditions.

FIG. 1

Polypeptide composition of X. laevis pol γ. Purified DNA pol γ was analyzed by SDS-PAGE and Coomassie blue staining. The solid arrowhead indicates the large subunit, and the open arrowhead indicates the small subunit. Numbers on the right indicate the molecular masses in kilodaltons of prestained protein mobility markers.

FIG. 2

X. laevis, Drosophila, and putative human pol γ small subunits (XLGB, GenBank accession no. AF124606; DMGB, accession no. U94702; and HSGB, accession no. U94703, respectively) were aligned with the glycyl-tRNA synthetase from T. thermophilus (TTGRS; accession no. P56206) by use of the program CLUSTAL from PCGene. Amino acids conserved among all four proteins are indicated by asterisks; similar amino acids are indicated by dots. Amino acids conserved between XLGB and TTGRS are indicated in bold. The three conserved motifs identified in class II glycyl-tRNA synthetases are also labeled under the sequences. Note that the X. laevis pol γB sequence begins with the N terminus of the mature protein and does not include the signal sequence.

FIG. 3

Copurification of large and small subunits of pol γ. Protein fractions collected during different steps of the purification of DNA pol γ were separated by SDS-PAGE and electroblotted onto PVDF membranes. The membranes were cut into upper and lower halves by use of prestained protein molecular weight markers as a guide. The upper part of each membrane was probed with an antibody directed against the DNA pol γ large subunit (L). The lower part of each membrane was probed with an antibody directed against the small subunit (S). Fractions are indicated by numbers, and FT denotes the flowthrough fraction. (A) Cation exchange (S Sepharose). (B) Gel filtration (Superdex 200 HiLoad). (C) Hydrophobic interaction (Poros PH).

FIG. 4

Characterization of recombinant pol γA and pol γB and analysis of primer-template binding by electrophoretic mobility shift assays (EMSA). (A) Xenopus pol γ (1,500 U) and recombinant human pol γA (180 U) were run in lanes 1 and 2, respectively, of an SDS–6% polyacrylamide gel, blotted to a PVDF membrane, and detected with a 1:20,000 dilution of antipeptide antibodies directed against the sequence TRRAVEPTWLTASN(C), contained in both human and Xenopus pol γA. (B) Coomassie blue-stain SDS-PAGE analysis of Xenopus pol γB purified from baculovirus-infected cells as described in Materials and Methods. Numbers at the right in panels A and B indicate the mobilities of protein mass standards in kilodaltons. (C) Autoradiogram of EMSA results. A 5′-³²P-labeled 44-mer hook oligonucleotide was incubated either alone (lane 1) or in the presence of either pol γ purified from X. laevis ovaries (lanes 2 and 6; labeled X) or recombinant subunits of pol γ, either human pol γA (lanes 3 and 7; labeled A), human pol γA plus Xenopus pol γB (lanes 4 and 8; labeled AB), or Xenopus pol γB alone (lanes 5 and 9; labeled B). Following incubation for 10 min at 25°C, affinity-purified antibodies raised against Xenopus DNA pol γB were added to reaction mixtures in lanes 6 through 9. Incubation was continued for an additional 10 min. Samples were subjected to electrophoresis under nondenaturing conditions, and ³²P radioactivity in the dried gel was detected by phosphorimager analysis.

FIG. 5

Stimulation of human DNA pol γA by Xenopus DNA pol γB. Polymerase activity of human DNA pol γA (●) and an equimolar mixture of human pol γA plus Xenopus pol γB (○) on oligo(dT)-poly(dA) was measured with reaction mixtures containing 2 (A) or 8 (B) mM MgCl₂. All reaction mixtures contained 0.2 pmol of the indicated polymerase subunits.

FIG. 6

Xenopus DNA pol γB confers increased processivity on human pol γA. Primer extension assays with singly primed M13 single-stranded DNA (A) or oligo(dT)-poly(dA) (B) were carried out as described in Materials and Methods. Each reaction included 200 fmol of annealed primer. In both panels, the reaction mixture in lane 1 contained no enzyme; those in lanes 2, 3, and 4 contained 75 U of Xenopus pol γ and were incubated for 1, 3, and 10 min, respectively. Reaction mixtures in lanes 5, 6, and 7 contained 9 U of recombinant human pol γA, equivalent to 50 fmol of polypeptide, and were incubated for the same periods of time; reactions in lanes 8, 9, and 10 were carried out with 50 fmol of recombinant human pol γA plus 50 fmol of recombinant Xenopus pol γB, incubated as in the previous reactions. Lane M shows DNA molecular weight markers of ³²P-labeled MspI fragments of plasmid pUC18, with the sizes, in nucleotides, indicated to the right of each panel. Due to space constraints, the larger marker fragments, of 501, 489, 404, and 353 nucleotides, are not individually labeled.