Characterization of the Native and Recombinant Catalytic Subunit of Human DNA Polymerase γ:  Identification of Residues Critical for Exonuclease Activity and Dideoxynucleotide Sensitivity

Abstract

The human DNA polymerase gamma catalytic subunit was overexpressed in recombinant baculovirus-infected insect cells, and the 136 000 Da protein was purified to homogeneity. Application of the same purification protocol to HeLa mitochondrial lysates permitted isolation of native DNA polymerase gamma as a single subunit, allowing direct comparison of the native and recombinant enzymes without interference of other polypeptides. Both forms exhibited identical properties, and the DNA polymerase and 3' --> 5' exonuclease activities were shown unambiguously to reside in the catalytic polypeptide. The salt sensitivity and moderate processivity of the isolated catalytic subunit suggest other factors could be required to restore the salt tolerance and highly processive DNA synthesis typical of gamma polymerases. To facilitate our understanding of mitochondrial DNA replication and mutagenesis as well as cytotoxicity mediated by antiviral nucleotide analogues, we also constructed two site-directed mutant proteins of the human DNA polymerase gamma. Substituting alanine for two essential acidic residues in the exonuclease motif selectively eliminated the 3' --> 5' exonucleolytic function of the purified mutant polymerase gamma. Replacement of a tyrosine residue critical for sugar recognition with phenylalanine in polymerase motif B reduced dideoxynucleotide inhibition by a factor of 5000 with only minor effects on overall polymerase function.

The Accessory Subunit of Xenopus laevis Mitochondrial DNA Polymerase γ Increases Processivity of the Catalytic Subunit of Human DNA Polymerase γ and Is Related to Class II Aminoacyl-tRNA Synthetases

“…In this experiment, we varied the monovalent salt concentration in reactions performed with either 2 or 8 mM MgCl 2 to encompass the range of physiological salt concentrations. Human pol ␥A was found to be very sensitive to increasing ionic strength when assayed at either 2 or 8 mM MgCl 2 , as reported previously for reactions performed with 1 mM MgCl 2 (19). The addition of the Xenopus small subunit markedly stimulated the activity of the human catalytic subunit at higher, more physiological KCl concentrations, but not under low-salt conditions.…”

“…As all our attempts to recover activity from the Xenopus recombinant catalytic subunit failed, we decided to determine whether the Xenopus small subunit would alter the properties of the active recombinant human catalytic subunit purified from insect cells (19). Figure 4A shows an immunoblot analysis of samples of Xenopus pol ␥ [lane 1; 1,500 U of enzyme, as assayed by TMP incorporation on poly(rA)-oligo(dT)] and recombinant human pol ␥A (lane 2; 180 U of enzyme, 14 ng of polypeptide).…”

“…The C-terminally His-tagged protein was purified by affinity chromatography on Ni-NTA superflow columns (Qiagen) with phosphate buffer under native conditions as recommended by the supplier, followed by chromatography on Mono S. The protein was concentrated by a second round of chromatography on Ni-NTA resin. The His-tagged recombinant catalytic subunit of human DNA pol ␥ was expressed and purified as described by Longley et al (19). To quantify the recombinant proteins, various quantities of the catalytic and accessory subunits were subjected to SDS-PAGE along with various amounts of quantitative protein standards (glutamate dehydrogenase and phosphorylase b; Boehringer Mannheim).…”

“…One published purification scheme for human DNA pol ␥ suggested the existence of a small subunit (11), but the potential relationship between this polypeptide and Drosophila pol ␥B has not been established. Recently, the catalytic subunit of human DNA pol ␥ was expressed in an active form (10,19). Surprisingly, the recombinant catalytic subunit alone displayed most of the characteristics of the enzyme purified from human cells, which did not appear to contain a stoichiometric amount of a small subunit.…”

Differential Incorporation and Removal of Antiviral Deoxynucleotides by Human DNA Polymerase γ

“…We and others have cloned and overproduced the catalytic subunit of human DNA polymerase ␥ in insect cells via a recombinant baculovirus (23)(24)(25). In this report, we have determined the insertion efficiency of the currently approved anti-HIV analogs into DNA by purified recombinant human DNA polymerase ␥, and we have investigated the efficiency of removing these analogs from DNA by the intrinsic 3Ј-5Ј exonuclease activity of pol ␥.…”